Treatment or prevention of cancer and precancerous disorders

ABSTRACT

The NSAID, sulindac and/or its metabolites and derivatives, in combination with hydrogen peroxide or another oxidizing agent, such as arsenic trioxide that generates reactive oxygen species (ROS), significantly enhances the killing of cancer cells. This effect occurs at concentrations of each compound that individually have little or no activity directed against cancer cells. A skin cream has been developed and used to treat skin cancer and precancerous skin growths that effectively removes the lesions with no effect on surrounding normal skin.

CROSS REFERENCE TO PRIOR APPLICATIONS

The present application is a continuation-in-part prosecution of U.S.application Ser. No. 11/388,248 filed Mar. 23, 2006 which claims thepriority of U.S. provisional patent application No. 60/664,383, entitled“TREATMENT OR PREVENTION OF CANCER AND PRECANCEROUS DISORDERS,” filedMar. 23, 2005. The foregoing are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

Compositions for treating cancer and methods of treating thereof. Inparticular, sulindac and/or its metabolites and derivatives, in eitherR- or S-epimeric form in combination with hydrogen peroxide or anotheroxidizing agent, such as arsenic trioxide that generates reactive oxygenspecies (ROS), significantly enhances the killing of cancer cells.

BACKGROUND OF THE INVENTION

Skin cancer is the most common form of cancer seen in the world. The twomost common types of non-melanoma skin cancer include basal cellcarcinoma (BCC) and squamous cell carcinoma (SCC). One in five Americanswill develop some form of skin cancer at some point in their lives, andit is estimated that over one million Americans will develop skin cancereach year.

Sun exposure has been implicated in the etiology of BCC and SCC. Theepidemic rise in the rate of these cancers is directly related toincreases in our population's outdoor activities and the desire for a“tanned” skin appearance. Pre-malignant actinic keratoses are commonskin growths induced by solar exposure that have the potential fordeveloping into SCC in upwards of 20% of cases. They often appear on theskin years before the development of cutaneous carcinomas.

It is considered a standard of care to remove as many skin cancers andactinic keratoses as possible with the least amount of discomfort,inconvenience and trauma (morbidity), for the patient. Destruction usingliquid nitrogen or electrodessication and curettage are effective inremoving a majority of skin cancers and actinic keratoses. However,these treatments may not be practical for certain skin cancers locatedon the face and extremities. Surgical removal of skin cancers andactinic keratoses is not always possible or desirable. Surgery is notpractical when many small actinic keratoses are present, and thescarring produced by surgery is generally unacceptable for exposed,relatively visible areas of the skin. Furthermore, it is believed that,in the early stages of their development, many skin cancers and actinickeratoses are so small that it would be difficult or impossible toremove surgically because they are not visible to the eye. Surgery,while necessary for the welfare of the patient, may place the patient atrisk and ultimately jeopardize their health if the cancer is locatedadjacent to certain vital areas, such as the eye. In addition, surgerymay lead to a poor cosmetic effect and leave the patient visiblydeformed.

Topically applied, chemical agents such as 5-fluorouracil (5-FU, Efudex,Fluoroplex), masoprocol (Actinex), imiquimod (Aldara), and diclofenac(Solaraze) have been approved to eradicate actinic keratoses. While 5-FUhas demonstrated efficacy for this purpose, it has been found to causepain, itching, skin inflammation, ulceration and cosmetic disfigurementoften so severe that patients hide at home and stop using it, thusmaking its therapeutic use unacceptable to many individuals. Theseeffects also preclude the use of 5-FU over large areas of the skin totreat incipient and/or microscopic actinic keratoses. Masoprocol wasremoved from the US market in 1996 after it was found to have a highincidence of contact sensitivity and allergic reactions. Imiquimod has arelatively good cosmetic effect when treating actinic keratoses but isvery expensive for use in large areas of the skin and its packaging inpouches has not been well received by many patients. The Food and DrugAdministration recently approved imiquimod for the treatment of BCC.However, this indication excludes treatment of BCC that occurs on theface. Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) usedto treat actinic keratoses. It has very modest effects and removal ofthe actinic keratoses may not be evident until months after treatmentends. However, it causes less irritation than 5-FU and imiquimod and maybe useful for some people.

There are several newer therapeutic approaches directed against actinickeratoses that are in clinical trials such as the use of photodynamictherapy (PDT) with aminolevulinic acid. This therapy is a two-steptreatment administered over a two-day period. First, the aminolevulinicacid is place over the lesion and on the next day, a blue light is usedto activate the drug. However, this treatment is expensive, needs to bedone at the doctor's office, is used only for thin lesions, and is notvery effective.

There is thus a need in the art to develop, safe, effective andspecificity of killing of abnormal cell, that is also cost effective.

SUMMARY

We have discovered that the NSAID, sulindac and/or its metabolites andderivatives, in combination with hydrogen peroxide or another oxidizingagent, such as arsenic trioxide that generates reactive oxygen species(ROS), significantly enhances the killing of cancer cells. This effectoccurs at concentrations of each compound that individually have littleor no activity directed against cancer cells. A skin cream has beendeveloped and used to treat skin cancer and precancerous skin growthsthat effectively removes the lesions with no effect on surroundingnormal skin.

We have shown that four tumor cell lines—two derived from skin (SCC andmelanoma), and colon and lung cell lines—exhibit markedly increasedsensitivities to killing by oxidative stress when pretreated withsulindac. Similar effects were observed with two different forms ofdirect oxidative stress—hydrogen peroxide and tert-butyl hydroperoxide(TBHP) or the use of arsenic trioxide, a compound that generates theproduction of ROS. This effect occurs at concentrations of each compoundthat individually have little or no activity directed against cancercells. The killing effect was not observed when using normal human skin,colon, or lung cells. In contrast, the normal cells exhibited aprotective effect with the combination of sulindac and peroxide at thelower concentrations of peroxide. Sulindac metabolites (sulindac sulfidein some instances and sulindac sulfone), and a sulindac derivative(sulindac methionine sulfoxide) also had enhanced killing effects ofcancer cells when exposed to peroxide. Experiments with four otherNSAIDS, (acetylsalicylic acid, ibuprofen, diclofenac sodium, andcelecoxib) indicated that none were effective in producing the enhancedkilling effects after exposure to the oxidating agents. The mechanism ofaction of this therapy may be associated with a selective increase inthe ROS levels in cancer cells as opposed to normal cells which, ingeneral, has an opposite effect leading to no change or a lowering inthe levels of ROS. Thus, this drug combination can be useful for thetreatment of cancer and precancerous conditions without the killing ofnormal cells.

When such combinations of sulindac and hydrogen peroxide are formulatedand applied topically using an inert vehicle to the area of the skinexhibiting the precancerous or cancerous condition, for example, anactinic keratosis, SCC, or a BCC, the lesions over a period of severalweeks completely resolve and disappear. There was minimal redness,swelling, and peeling with no itching, burning, sores or blisters thatdeveloped. An enhanced yellowish color was visibly present on the skincondition being treated prior to its destruction. This yellow color maybe an indication of patient compliance for the accurate documentation ofits clinical use by the patient. Sulindac or peroxide when applied aloneto the skin condition had no effect on the precancerous or cancerousgrowth.

It will thus be appreciated that there is a need for topically activeagents which are effective in removing actinic keratoses, BCC and SCC.These agents should attempt to avoid the undesirable side effects ofcurrent treatments, shorten the time for removal and healing of thelesions, lower the expense of the therapy, and increase its compliancefor use by the patient.

In a preferred embodiment, a pharmaceutical composition comprisessulindac, sulindac metabolites, sulindac derivatives and an oxidatingagent. Preferably, the sulindac metabolite comprises sulindac sulfide orsulindac sulfone. Preferably, the sulindac derivative is sulindacmethionine sulfoxide.

In another preferred embodiment, the sulindac comprises R- and S-epimersof sulindac. Preferably, the sulindac is an R-epimer. We have discoveredthat the R-epimer of sulindac can retain its cancer killing effects. Wehave also found that in normal cells, but not cancer cells, the R-epimerof sulindac is not efficiently converted to the sulfide, the active COXinhibitor. Since COX inhibitors produce serious toxicity, it is expectedthat the R-epimer of sulindac can be a potentially superior therapeuticagent because it would have a lower toxicity profile in normal cells.

In another preferred embodiment, the oxidating agent comprises at leastone of: peroxides, nitrates, nitrites, perchlorates, chlorates,chlorites, hypochlorite, dichromates, permanganates, and persulfates,bromine, arsenic trioxide, retinoic acid (and its derivatives), Kantimonyl tartrate, doxorubicin, imexon, and bortezomib, hydrogenperoxide; inorganic peroxides, sodium peroxyborate tetrahydrate (sodiumperborate tetrahydrate) calcium peroxide; peroxide complexes, ureahydrogen peroxide; superoxide salts, sodium superoxide; superoxide freeradical (O₂*) dismutates; organic peroxides, hydroperoxides (ROOH),lipid hydroperoxides, artemisinin and derivatives thereof;1-Hydroperoxycyclohexyl-1-hydroxy cyclohexyl peroxide, tert-Butylhydroperoxide; fatty acid hydroperoxides, linolenic acid hydroperoxide,arachidonic acid hydroperoxide, docosahexaenoic acid hydroperoxide,cholesterol hydroperoxide, cholesteryl linoleate hydroperoxide,trilinolein hydroperoxide, phosphatidylcholine hydroperoxide,phosphatidylethanolamine hydroperoxide; 3-chloroperoxybenzoic acid,1,1-bis(tert-butylperoxy)cyclohexane, peracetic acid, monoperoxyphthalicacid, tert-butyl peroxide,2,5,bis(tert-butylperoxy)-2,5-dimethyl-3-hexyne, cumene hydroperoxide,2,5-bis(tert-butylperoxy)-2,5-dimethylhexane, lauroyl peroxide, benzoylperoxide, dicumyl peroxide, 2,5-dihydroperoxy-2,5-dimethylhexane,tert-butyl peracetate, tert-amyl hydroperoxide, diisononanoyl peroxide,decanoyl peroxide, succinic acid peroxide, 2,4-pentanedione peroxide,di-tert-butyl peroxide, tert-butyl cumyl peroxide,bis(t-butylperoxy)-diisopropylbenzene, and1-((hydroperoxycyclohexyl)dioxy)-cyclohexanol; hydrogen peroxide,compounds containing a peroxy (peroxo) —O—O— moiety, superoxides,peroxide precursor compounds; endoperoxides, diacyl peroxides, ketoneperoxides, peroxydicarbonates, peroxyesters, dialkyl peroxides,peroxyketals, and peroxyacids. Preferably, the oxidating agent ishydrogen peroxide, and/or tert-butyl hydroperoxide (TBHP), and/orarsenic trioxide.

In another preferred embodiment, the oxidizing agent is hydrogenperoxide in a concentration is in a range of about 1% to 50% by weight.

In another preferred embodiment, the sulindac concentration is in rangefrom about 1% to 50% by weight.

In yet another embodiment, the oxidizing agent is arsenic trioxide in aconcentration in a range of about 1% to 50% by weight.

In another preferred embodiment, a topical pharmaceutical compositioncomprises a 5% sulindac gel and an oxidizing agent. Preferably, the 5%sulindac gel comprises: 71.08% deionized water; 12.00% SD Alcohol 40;10% KOH; 5% Sulindac; 1% Hydroxy methyl cellulose; 0.50% Xantham gum;0.20% Glydant Plus; 0.20% Citric Acid; and 0.02% disodium EDTA.

In other preferred embodiments, the sulindac gel is 10% sulindac, 15%sulindac, 20% sulindac, 50% sulindac.

In another preferred embodiment, a method of treating an abnormal cell,said method comprising administering to an abnormal cell apharmaceutical composition comprising sulindac, or sulindac metabolites,or sulindac derivatives or combinations thereof, and an oxidating agent,thereby treating an abnormal cell. Preferably, the sulindac metabolitecomprises sulindac sulfide or sulindac sulfone.

In another preferred embodiment, the oxidating agent comprises at leastone of: peroxides, nitrates, nitrites, perchlorates, chlorates,chlorites, hypochlorite, dichromates, permanganates, and persulfates,bromine, arsenic trioxide, retinoic acid (and its derivatives), Kantimonyl tartrate, doxorubicin, imexon, and bortezomib, hydrogenperoxide; inorganic peroxides, sodium peroxyborate tetrahydrate (sodiumperborate tetrahydrate) calcium peroxide; peroxide complexes, ureahydrogen peroxide; superoxide salts, sodium superoxide; superoxide freeradical (O₂*) dismutates; organic peroxides, hydroperoxides (ROOH),lipid hydroperoxides, artemisinin and derivatives thereof;1-Hydroperoxycyclohexyl-1-hydroxy cyclohexyl peroxide, tert-Butylhydroperoxide; fatty acid hydroperoxides, linolenic acid hydroperoxide,arachidonic acid hydroperoxide, docosahexaenoic acid hydroperoxide,cholesterol hydroperoxide, cholesteryl linoleate hydroperoxide,trilinolein hydroperoxide, phosphatidylcholine hydroperoxide,phosphatidylethanolamine hydroperoxide; 3-chloroperoxybenzoic acid,1,1-bis(tert-butylperoxy)cyclohexane, peracetic acid, monoperoxyphthalicacid, tert-butyl peroxide,2,5,bis(tert-butylperoxy)-2,5-dimethyl-3-hexyne, cumene hydroperoxide,2,5-bis(tert-butylperoxy)-2,5-dimethylhexane, lauroyl peroxide, benzoylperoxide, dicumyl peroxide, 2,5-dihydroperoxy-2,5-dimethylhexane,tert-butyl peracetate, tert-amyl hydroperoxide, diisononanoyl peroxide,decanoyl peroxide, succinic acid peroxide, 2,4-pentanedione peroxide,di-tert-butyl peroxide, tert-butyl cumyl peroxide,bis(t-butylperoxy)-diisopropylbenzene, and1-((hydroperoxycyclohexyl)dioxy)-cyclohexanol; hydrogen peroxide,compounds containing a peroxy (peroxo) —O—O— moiety, superoxides,peroxide precursor compounds; endoperoxides, diacyl peroxides, ketoneperoxides, peroxydicarbonates, peroxyesters, dialkyl peroxides,peroxyketals, and peroxyacids. Preferably, the oxidating agent ishydrogen peroxide and/or tert-butyl hydroperoxide (TBHP), and/or arsenictrioxide.

In another preferred embodiment, the oxidizing agent, hydrogen peroxide,concentration is in a range of about 1% to 50% by weight.

In another preferred embodiment, the sulindac concentration is in rangefrom about 1% to 50% by weight.

In another preferred embodiment, the oxidizing agent, arsenic trioxide,concentration is in a range of about 1% to 50% by weight.

In another preferred embodiment, the pharmaceutical compositioncomprises a 5% sulindac gel and an oxidizing agent. Preferably, the 5%sulindac gel comprises: 71.08% deionized water; 12.00% SD Alcohol 40;10% KOH; 5% Sulindac; 1% Hydroxy methyl cellulose; 0.50% Xantham gum;0.20% Glydant Plus; 0.20% Citric Acid; and 0.02% disodium EDTA.

In other preferred embodiments, the sulindac gel is 10% sulindac, 15%sulindac, 20% sulindac, 50% sulindac.

In yet another embodiment, a method of treating cancer, said methodcomprising administering to a cancer cell a pharmaceutical compositioncomprising sulindac, or sulindac metabolites, or sulindac derivatives orcombinations thereof, and an oxidating agent, thereby treating cancer.Sulindac, sulindac metabolites, sulindac derivatives and an oxidatingagent. Preferably, the sulindac metabolite comprises sulindac sulfide orsulindac sulfone.

In another preferred embodiment, the oxidating agent comprises at leastone of: peroxides, nitrates, nitrites, perchlorates, chlorates,chlorites, hypochlorite, dichromates, permanganates, and persulfates,bromine, arsenic trioxide, retinoic acid (and its derivatives), Kantimonyl tartrate, doxorubicin, imexon, and bortezomib, hydrogenperoxide; inorganic peroxides, sodium peroxyborate tetrahydrate (sodiumperborate tetrahydrate) calcium peroxide; peroxide complexes, ureahydrogen peroxide; superoxide salts, sodium superoxide; superoxide freeradical (O₂*) dismutates; organic peroxides, hydroperoxides (ROOH),lipid hydroperoxides, artemisinin and derivatives thereof;1-Hydroperoxycyclohexyl-1-hydroxy cyclohexyl peroxide, tert-Butylhydroperoxide; fatty acid hydroperoxides, linolenic acid hydroperoxide,arachidonic acid hydroperoxide, docosahexaenoic acid hydroperoxide,cholesterol hydroperoxide, cholesteryl linoleate hydroperoxide,trilinolein hydroperoxide, phosphatidylcholine hydroperoxide,phosphatidylethanolamine hydroperoxide; 3-chloroperoxybenzoic acid,1,1-bis(tert-butylperoxy)cyclohexane, peracetic acid, monoperoxyphthalicacid, tert-butyl peroxide,2,5,bis(tert-butylperoxy)-2,5-dimethyl-3-hexyne, cumene hydroperoxide,2,5-bis(tert-butylperoxy)-2,5-dimethylhexane, lauroyl peroxide, benzoylperoxide, dicumyl peroxide, 2,5-dihydroperoxy-2,5-dimethylhexane,tert-butyl peracetate, tert-amyl hydroperoxide, diisononanoyl peroxide,decanoyl peroxide, succinic acid peroxide, 2,4-pentanedione peroxide,di-tert-butyl peroxide, tert-butyl cumyl peroxide,bis(t-butylperoxy)-diisopropylbenzene, and1-((hydroperoxycyclohexyl)dioxy)-cyclohexanol; hydrogen peroxide,compounds containing a peroxy (peroxo) —O—O— moiety, superoxides,peroxide precursor compounds; endoperoxides, diacyl peroxides, ketoneperoxides, peroxydicarbonates, peroxyesters, dialkyl peroxides,peroxyketals, and peroxyacids. Preferably, the oxidating agent ishydrogen peroxide, and/or tert-butyl hydroperoxide (TBHP), and/orarsenic trioxide.

In another preferred embodiment, the oxidizing agent is hydrogenperoxide in a concentration is in a range of about 1% to 50% by weight.

In another preferred embodiment, the sulindac concentration is in rangefrom about 1% to 50% by weight.

In yet another embodiment, the oxidizing agent is arsenic trioxide in aconcentration in a range of about 1% to 50% by weight.

In another preferred embodiment, a topical pharmaceutical compositioncomprises a 5% sulindac gel and an oxidizing agent. Preferably, the 5%sulindac gel comprises: 71.08% deionized water; 12.00% SD Alcohol 40;10% KOH; 5% Sulindac; 1% Hydroxy methyl cellulose; 0.50% Xantham gum;0.20% Glydant Plus; 0.20% Citric Acid; and 0.02% disodium EDTA.

In other preferred embodiments, the sulindac gel is 10% sulindac, 15%sulindac, 20% sulindac, 50% sulindac. The oxidizing agent comprisesbetween about 1% to 50% of the formulation. The oxidizing agent can be,for example, hydrogen peroxide. In one aspect, the sulindac gelformulation comprises 10% sulindac and 25% hydrogen peroxide.

Other aspects of the invention are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is pointed out with particularity in the appended claims.The above and further advantages of this invention may be betterunderstood by referring to the following description taken inconjunction with the accompanying drawings, in which:

FIG. 1 is a graph showing the effect of TBHP on viability of SCC skincancer cells following pretreatment with 500 μM sulindac. SCC skincancer cells were incubated for 24 hr in the presence or absence of 500μM sulindac. The medium was removed and the cells washed once with freshmedium followed by addition of medium containing the indicatedconcentration of TBHP. After a 2 hr incubation at 37° C., the cells wereassayed for viability.

FIG. 2 is a graph showing the effect of hydrogen peroxide on viabilityof SCC skin cancer cells following pretreatment with 500 μM sulindac.SCC skin cancer cells were incubated for 24 hr in the presence orabsence of 500 μM sulindac. The medium was removed and the cells washedonce with fresh medium followed by addition of medium containing theindicated concentration of hydrogen peroxide (H₂O₂). After a 24 hrincubation at 37° C., the cells were assayed for viability as describedin the text.

FIG. 3 is a graph showing the effect of arsenic trioxide on viability ofSCC skin cancer cells following pretreatment with 500 μM sulindac. SCCskin cancer cells were incubated for 24 hr in the presence or absence of500 μM sulindac. The medium was removed and the cells washed once withfresh medium followed by addition of medium containing the indicatedconcentration of arsenic trioxide. After a 24 hr incubation at 37° C.,the cells were assayed for viability as described in the text.

FIG. 4 is a graph showing the effect of TBHP on viability of RKO coloncancer cells following pretreatment with 500 μM sulindac. RKO coloncancer cells were incubated for 24 hr in the presence (▪) or absence (♦)of 500 μM sulindac. The medium was removed and the cells washed oncewith fresh medium followed by addition of medium containing theindicated concentration of TBHP. After a 2 hr incubation at 37° C., thecells were assayed for viability as described in the text. Error barsrepresent the SEM of four replicate samples.

FIG. 5 is a graph showing the effect of hydrogen peroxide on viabilityof RKO colon cancer cells following pretreatment with 500 μM sulindac.RKO colon cancer cells were incubated for 24 hr in the presence (▪) orabsence (♦) of 500 μM sulindac. The medium was removed and the cellswashed once with fresh medium followed by addition of medium containingthe indicated concentration of hydrogen peroxide (H₂O₂). After a 2 hrincubation at 37° C., the cells were assayed for viability as describedin the text.

FIG. 6 is a graph showing the effect of TBHP on viability of lung cancercells following pretreatment with 500 μM sulindac. Lung cancer cellswere incubated for 24 hr in the presence (▪) or absence (♦) of 500 μMsulindac. The medium was removed and the cells washed once with freshmedium followed by addition of medium containing the indicatedconcentration of TBHP. After a 2 hr incubation at 37° C., the cells wereassayed for viability as described in the text. Error bars represent theSEM of four replicate samples.

FIG. 7 is a graph showing the effect of hydrogen peroxide on viabilityof lung cancer cells following pretreatment with 500 μM sulindac. Lungcancer cells were incubated for 24 hr in the presence (▪) or absence (♦)of 500 μM sulindac. The medium was removed and the cells washed oncewith fresh medium followed by addition of medium containing theindicated concentration of hydrogen peroxide. After a 2 hr incubation at37° C., the cells were assayed for viability as described in the text.Error bars represent the SEM for replicate samples.

FIG. 8 is a graph showing the effect of TBHP on viability of melanomacells following pretreatment with 500 μM sulindac. Melanoma skin cancercells were incubated for 24 hr in the presence or absence of 500 μMsulindac. The medium was removed and the cells washed once with freshmedium followed by addition of medium containing the indicatedconcentration of TBHP. After a 2 hr incubation at 37° C., the cells wereassayed for viability as described in the text.

FIG. 9 is a graph effect of arsenic trioxide on viability of melanomacells following pretreatment with 500 μM sulindac. Melanoma cells wereincubated for 24 hr in the presence or absence of 500 μM sulindac. Themedium was removed and the cells washed once with fresh medium followedby addition of medium containing the indicated concentration of arsenictrioxide. After a 24 hr incubation at 37° C., the cells were assayed forviability as described in the text.

FIGS. 10A and 10 B are graphs showing the effect of TBHP on viability ofnormal human epidermal keratinocytes following pretreatment with 500 μMsulindac. Normal human epidermal keratinocyte cells were incubated for24 hr in the presence or absence of 500 μM sulindac. The medium wasremoved and the cells washed once with fresh medium followed by additionof medium containing the indicated concentration of TBHP (FIG. 10A) orarsenic trioxide (FIG. 10B). After a 2 hr incubation with TBHP or a24-hour incubation with arsenic trioxide at 37° C., the cells wereassayed for viability as described in the text.

FIG. 11 is a graph showing the effect of TBHP on normal human coloncells pretreated with sulindac. Normal diploid colon cells wereincubated for 24 hr with medium containing 500 μM sulindac or noaddition (no sulindac). Cells were washed free of sulindac prior toincubation for 2 hr with the indicated concentration of TBHP. Cellviability was measured using the MTS assay described. Results areexpressed as % of Control (cells that were not pretreated or exposed toTBHP). Error bars are standard error of the mean (SEM) expressed as a %of the mean value of four replicate samples.

FIG. 12 is a graph showing the effect of TBHP on normal human lung cellspretreated with sulindac. Normal diploid lung cells were incubated for24 hr with medium containing 500 μM sulindac or no addition (nosulindac). Cells were washed free of sulindac prior to incubation for 2hr with the indicated concentration of TBHP. Cell viability was measuredusing the MTS assay. Results are expressed as % of Control (cells thatwere not pretreated or exposed to TBHP). Error bars are standard errorof the mean (SEM) expressed as a % of the mean value of four replicatesamples.

FIGS. 13A-D shows the structures of (FIG. 13A) Sulindac (FIG. 13B)Sulindac Sulfide (FIG. 13C) Sulindac Methionine Sulfoxide and D)Sulindac Sulfone.

FIG. 14 is a graph showing skin cancer cells treated with sulindac,sulindac metabolites, or a sulindac derivative. Skin cancer cells wereincubated for 24 hr with medium containing 500 μM sulindac, 500 μMsulindac sulfone, 500 μM SMO, 50 μM sulindac sulfide or no addition (nopretreatment). Cells were washed free of compound prior to incubationfor 2 hr with the indicated concentration of TBHP. Cell viability wasmeasured using the MTS assay.

FIG. 15 is a graph showing the effect of sulindac and its metabolites onthe viability of lung cancer cells in response to oxidative stress. Lungcancer cells were incubated for 24 hr with medium containing 500 μMsulindac (▪), 250 μM sulindac sulfone (▴), 250 μM sulindac sulfide ( )or no addition (♦). For all concentrations of TBHP tested, p<0.05.

FIG. 16 is a graph showing a constant non-toxic TBHP concentration incombination with escalating doses of sulindac, the effects on normal vs.cancer cells can be better elucidated. There is a protective effect onnormal cells while an enhanced killing of cancer cells occurs. In thisexperiment, the gap or difference in cell survival widens between thecell types over a 3-5× concentration range of sulindac (500 μM-1500 μM).

FIG. 17 is a scan of a photograph showing basal cell carcinomas beforetreatment with sulindac peroxide. The black arrows point to tworecurrent basal cell carcinomas on the face of a patient. A 0.5centimeter ulcerated area with pearly irregular borders on the rightforehead near the right upper eyelid and a 1.5 centimeter poorlycircumscribed lesion with pearly ill-defined nodular borders on theright temple area near the hairline.

FIG. 18 is a scan of a photograph showing the basal cell carcinomas ofFIG. 17, after treatment with sulindac peroxide. The black arrows pointto the two areas on the skin of the face that previously containedbiopsy proven recurrent basal cell skin cancers (as described in FIG.17) showing the disappearance of the cancers after treatment withsulindac-peroxide formulations. The areas have healed with small amountsof scar tissue present.

FIG. 19 is a scan of a photograph showing a hyperkeratotic lesion beforetreatment with the sulindac-peroxide formulation. The white arrow pointsto a 1 centimeter hyperkeratotic lesion on the right hand representing ahypertrophic actinic keratosis or squamous cell carcinoma beforetreatment with sulindac-peroxide formulations.

FIG. 20 is a scan of a photograph showing the hyperkeratotic lesionafter treatment with sulindac-peroxide of the same patient in FIG. 19.The white arrow points to the area of previous skin lesion (as describedin FIG. 19) showing the eradication of the condition after treatmentwith the sulindac-peroxide formulations. Only a small amount of scartissue with minimal scaling remains.

FIG. 21 is a schematic representation showing the structure of sulindacand its metabolites. Sulindac refers to a mixture of R- and S-epimers ofsulindac sulfoxide. Theoretically, both the R-sulindac sulfoxide andS-sulindac sulfoxide can be oxidized to form sulindac sulfone or reducedto form sulindac sulfide.

FIG. 22 is a chromatogram showing the separation of the R&S-epimers ofsulindac using a chiral column. Sulindac (7 mg/ml) was dissolved in amixture of hexane/ethanol (65/35) containing 0.1% acetic acid and 75 μlof this solution applied to the chiral column. The column was developedwith the above solvent at a flow rate of 1.5 ml/min and 300 μl fractionscollected. The elution of the sulindac was followed by monitoring theoptical density at 256 nm. Two peaks are observed eluting after 22.5 and28 minutes (OD 256). The R-form elutes first (22.5 min) followed by theS-form (28 min).

FIG. 23 is a graph showing the metabolic conversion of the R & S epimersof sulindac to the sulfide form in cardiac myocytes. The amount ofsulindac sulfide formed by 10⁶ cells incubated with 400 μM sulindac, R,S or RS mixture is measured as a function of time of incubation. Theassay was performed by HPLC separation of cell lysates, using a C18column and a mobile phase of 50/50% 50 mM sodium acetate, pH 4.73, andacetonitrile. The absorbance was measured at 330 nm.

FIG. 24 is a graph showing the effect of R- & S-sulindac epimers on thekilling of SCC cells after exposure to TBHP. SCC skin cancer cells wereincubated for 24 hr in the presence or absence of 500 μM sulindac or 500μM sulindac-R or -S epimers. The medium was removed and the cells washedonce with fresh medium followed by addition of medium containing theindicated concentration of TBHP. After a 2 hr incubation at 37° C., thecells were assayed for viability as described in the text.

FIG. 25 is a graph showing the effect of R- & S-sulindac epimers on thekilling of lung cancer cells after exposure to hydrogen peroxide. Lungcancer cells were treated with 500 μM Sulindac-R, 500 μM Sulindac-S or500 μM Sulindac-(R+S) for 24 hours. The cells were washed free of thesulindac before incubation for 24-hrs with the indicated concentrationof hydrogen peroxide. Cell viability was measured using the MTS assay asdescribed in the text. The error bars are the SEM for four replicatesamples.

FIG. 26 is a graph showing lung cancer cells treated with 500 μMsulindac-R, 500 μM sulindac-S or 500 μM sulindac-(R+S) for 24 hr. Thecells were then washed free of the sulindac before exposure to hydrogenperoxide over a range of 0-4 mM. Viability was measured by the MTSassay. Enhanced killing of the cancer cells is over the range of 1.5-3.0mM hydrogen peroxide.

DETAILED DESCRIPTION

Treatment or prevention of a cancerous or precancerous disorder inhumans or animals in need of such treatment or prevention is provided bymethods and combinations using two or more components.

DEFINITIONS

In accordance with the present invention and as used herein, thefollowing terms are defined with the following meanings, unlessexplicitly stated otherwise.

As used herein, “a”, “an,” and “the” include plural references unlessthe context clearly dictates otherwise.

As used herein, “cancer” refers to all types of cancer or neoplasm ormalignant tumors found in mammals, including, but not limited to:leukemias, lymphomas, melanomas, carcinomas and sarcomas. Examples ofcancers are cancer of the brain, breast, pancreas, cervix, colon, head &neck, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary,sarcoma, stomach, uterus and Medulloblastoma. The term “cancer” includesany cancer arising from a variety of chemical, physical, infectiousorganism cancer causing agents. For example, hepatitis B virus,hepatitis C virus, human papillomaviruses; sun; lead and lead compounds,X-rays, compounds found in grilled meats, and a host of substances usedin textile dyes, paints and inks. Further details of cancer causingagents are listed in The Report on Carcinogens, Eleventh Edition.Federal law requires the Secretary of the Department of Health and HumanServices to publish the report every two years.

Additional cancers which can be treated by the disclosed compositionaccording to the invention include but not limited to, for example,Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma,neuroblastoma, breast cancer, ovarian cancer, lung cancer,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,small-cell lung tumors, primary brain tumors, stomach cancer, coloncancer, malignant pancreatic insulanoma, malignant carcinoid, urinarybladder cancer, premalignant skin lesions, testicular cancer, lymphomas,thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tractcancer, malignant hypercalcemia, cervical cancer, endometrial cancer,adrenal cortical cancer, and prostate cancer.

The term “sarcoma” generally refers to a tumor which is made up of asubstance like the embryonic connective tissue and is generally composedof closely packed cells embedded in a fibrillar or homogeneoussubstance. Examples of sarcomas which can be treated with thecompositions and optionally a potentiator and/or chemotherapeutic agentinclude, but not limited to a chondrosarcoma, fibrosarcoma,lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy'ssarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma,ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma,stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma,giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathicmultiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of Bcells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma,Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma,malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocyticsarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, andtelangiectaltic sarcoma.

The term “melanoma” is taken to mean a tumor arising from themelanocytic system of the skin and other organs. Melanomas which can betreated with the compositions and optionally a potentiator and/oranother chemotherapeutic agent include but not limited to, for example,acral-lentiginous melanoma, amelanotic melanoma, benign juvenilemelanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma,juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodularmelanoma, subungal melanoma, and superficial spreading melanoma.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Carcinomas which can be treated with thecompositions and optionally a potentiator and/or a chemotherapeuticagent include but not limited to, for example, acinar carcinoma, acinouscarcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinomaadenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolarcell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloidcarcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma,bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma,cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma,comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma encuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cellcarcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma,encephaloid carcinoma, epiermoid carcinoma, carcinoma epithelialeadenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum,gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma,carcinoma gigantocellulare, glandular carcinoma, granulosa cellcarcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellularcarcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroidcarcinoma, infantile embryonal carcinoma, carcinoma in situ,intraepidermal carcinoma, intraepithelial carcinoma, Krompecher'scarcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticularcarcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelialcarcinoma, carcinoma medullare, medullary carcinoma, melanoticcarcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum,carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum,mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oatcell carcinoma, carcinoma ossificans, osteoid carcinoma, papillarycarcinoma, periportal carcinoma, preinvasive carcinoma, prickle cellcarcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reservecell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma,scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma,carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidalcell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamouscarcinoma, squamous cell carcinoma, string carcinoma, carcinomatelangiectaticum, carcinoma telangiectodes, transitional cell carcinoma,carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, andcarcinoma villosum.

“Diagnostic” or “diagnosed” means identifying the presence or nature ofa pathologic condition or a patient susceptible to a disease. Diagnosticmethods differ in their sensitivity and specificity. The “sensitivity”of a diagnostic assay is the percentage of diseased individuals who testpositive (percent of “true positives”). Diseased individuals notdetected by the assay are “false negatives.” Subjects who are notdiseased and who test negative in the assay, are termed “truenegatives.” The “specificity” of a diagnostic assay is 1 minus the falsepositive rate, where the “false positive” rate is defined as theproportion of those without the disease who test positive. While aparticular diagnostic method may not provide a definitive diagnosis of acondition, it suffices if the method provides a positive indication thataids in diagnosis.

The terms “patient” or “individual” are used interchangeably herein, andrefers to a mammalian subject to be treated, with human patients beingpreferred. In some cases, the methods of the invention find use inexperimental animals, in veterinary application, and in the developmentof animal models for disease, including, but not limited to, rodentsincluding mice, rats, and hamsters; and primates.

As used herein, a “pharmaceutically acceptable” component is one that issuitable for use with humans and/or animals without undue adverse sideeffects (such as toxicity, irritation, and allergic response)commensurate with a reasonable benefit/risk ratio.

As used herein, the term “safe and effective amount” refers to thequantity of a component which is sufficient to yield a desiredtherapeutic response without undue adverse side effects (such astoxicity, irritation, or allergic response) commensurate with areasonable benefit/risk ratio when used in the manner of this invention.By “therapeutically effective amount” is meant an amount of a compoundof the present invention effective to yield the desired therapeuticresponse. For example, an amount effective to delay the growth of or tocause a cancer, either a sarcoma or lymphoma, or to shrink the cancer orprevent metastasis. The specific safe and effective amount ortherapeutically effective amount will vary with such factors as theparticular condition being treated, the physical condition of thepatient, the type of mammal or animal being treated, the duration of thetreatment, the nature of concurrent therapy (if any), and the specificformulations employed and the structure of the compounds or itsderivatives.

As used herein, a “pharmaceutical salt” include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids. Preferablythe salts are made using an organic or inorganic acid. These preferredacid salts are chlorides, bromides, sulfates, nitrates, phosphates,sulfonates, formates, tartrates, maleates, malates, citrates, benzoates,salicylates, ascorbates, and the like. The most preferred salt is thehydrochloride salt.

“Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology or symptoms of adisorder. Accordingly, “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures. “Treatment” may also bespecified as palliative care. Those in need of treatment include thosealready with the disorder as well as those in which the disorder is tobe prevented. In tumor (e.g., cancer) treatment, a therapeutic agent maydirectly decrease the pathology of tumor cells, or render the tumorcells more susceptible to treatment by other therapeutic agents, e.g.,radiation and/or chemotherapy.

The treatment of neoplastic disease, cancer, or neoplastic cells, refersto an amount of the composition, described throughout the specificationand in the Examples which follow, capable of invoking one or more of thefollowing effects: (1) inhibition, to some extent, of tumor growth,including, (i) slowing down and (ii) complete growth arrest; (2)reduction in the number of tumor cells; (3) maintaining tumor size; (4)reduction in tumor size; (5) inhibition, including (i) reduction, (ii)slowing down or (iii) complete prevention of tumor cell infiltrationinto peripheral organs; (6) inhibition, including (i) reduction, (ii)slowing down or (iii) complete prevention of metastasis; (7) enhancementof anti-tumor immune response, which may result in (i) maintaining tumorsize, (ii) reducing tumor size, (iii) slowing the growth of a tumor,(iv) reducing, slowing or preventing invasion or (v) reducing, slowingor preventing metastasis; and/or (8) relief, to some extent, of one ormore symptoms associated with the disorder.

The terms “dosing” and “treatment” as used herein refer to any process,action, application, therapy or the like, wherein a subject,particularly a human being, is rendered medical aid with the object ofimproving the subject's condition, either directly or indirectly.

The term “therapeutic compound” as used herein refers to a compounduseful in the prophylaxis or treatment of cancer.

The term “therapeutic combination” as used herein refers to theadministered therapeutic compounds when administered in combinationtherapy, and to any pharmaceutically acceptable carriers used to providedosage forms such that the beneficial effect of each therapeuticcompound is realized by the subject at the desired time, whether thecompounds are administered substantially simultaneously, orsequentially.

The compounds of the invention encompass various isomeric forms. Suchisomers include, e.g., stereoisomers, e.g., chiral compounds, e.g.,diastereomers and enantiomers.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “diastereomers” refers to stereoisomers with two or morecenters of dissymmetry and whose molecules are not mirror images of oneanother.

The term “enantiomers” refers to two stereoisomers of a compound whichare non-superimposable mirror images of one another. An equimolarmixture of two enantiomers is called a “racemic mixture” or a“racemate.”

The term “isomers” or “stereoisomers” refers to compounds which haveidentical chemical constitution, but differ with regard to thearrangement of the atoms or groups in space. Diastereoisomers that havethe opposite configuration at only one of two or more tetrahedralstereogenic centres present in the respective molecular entities areknown as “epimers.” Thus, when used herein “sulindac” or variants,derivatives or oxides thereof, includes epimeric and enantiomericmolecules.

Furthermore the indication of configuration across a carbon-carbondouble bond can be “Z” referring to what is often referred to as a “cis”(same side) conformation whereas “E” refers to what is often referred toas a “trans” (opposite side) conformation. Regardless, bothconfigurations, cis/trans and/or Z/E are contemplated for the compoundsfor use in the present invention.

With respect to the nomenclature of a chiral center, the terms “d” and“1” configuration are as defined by the IUPAC Recommendations. As to theuse of the terms, diastereomer, racemate, epimer and enantiomer, thesewill be used in their normal context to describe the stereochemistry ofpreparations.

Compositions

The present invention provides a composition comprising sulindac andhydrogen peroxide which effectively removes actinic keratoses, BCC, andSCC in several weeks without severe side effects. The pharmaceuticalcomposition may be applied to specific sites or to relatively largeareas of skin to treat visible actinic keratoses and skin cancers and toprevent the development of incipient actinic keratoses. The compositioncan be used simultaneously or in separate parts. The advantages of thepresent invention will be readily apparent from the discussion,description and examples which follow.

Briefly, we have shown that four tumor cell lines—two derived from skin(SCC and melanoma), and colon and lung cell lines—exhibit markedlyincreased sensitivities to killing by oxidative stress when pretreatedwith sulindac. Similar effects were observed with two different forms ofdirect oxidative stress—hydrogen peroxide and tert-butyl hydroperoxide(TBHP) or the use of arsenic trioxide, a compound that generates theproduction of ROS. This effect occurs at concentrations of each compoundthat individually have little or no activity directed against cancercells. The killing effect was not observed when using normal human skin,colon, or lung cells. In contrast, the normal cells exhibited aprotective effect with the combination of sulindac and peroxide at thelower concentrations of peroxide. Sulindac metabolites (sulindac sulfidein some instances and sulindac sulfone) also had enhanced killingeffects of cancer cells when exposed to peroxide. Experiments with fourother NSAIDS, (acetylsalicylic acid, ibuprofen, diclofenac sodium, andcelecoxib) indicated that none were effective in producing the enhancedkilling effects after exposure to the oxidating agents. The mechanism ofaction of this therapy may be associated with a selective increase inthe ROS levels in cancer cells as opposed to normal cells which, ingeneral, has an opposite effect leading to no change or a lowering inthe levels of ROS. Thus, this drug combination can be useful for theselective treatment of cancer and precancerous conditions without thekilling of normal cells.

The method comprises treatment with a therapeutically effective amountof a combination comprising two agents. The first agent is sulindac orits metabolites or derivatives. The second agent is an oxidizing agentor agent that leads to the generation of ROS. Besides being useful forhuman treatment, the present invention is also useful for veterinarytreatment of companion animals, exotic animals and farm animals,including mammals, rodents, and the like. More preferred animals includehorses, dogs, and cats.

The enhanced cancer cell killing effects of the combination of sulindacand an oxidizing agent has not been previously described. Withoutwishing to be bound by theory, sulindac functions as a catalyticanti-oxidant through the Msr system, thereby leading to an enhancedprotection of cells (normal or cancerous) when given in combination withan oxidant.

The experimental results revealed that sulindac had a dual effect, in asense a reverse effect, such that normal cells were protected but cancercells were killed. This effect is specific to sulindac and is not seenwith other NSAIDs. The mechanism of action suggests that cancer cells orhighly proliferating cells, become more sensitive to the effects of ROS.Normal cells or cells that are not in a hyper-proliferative state areprotected and become less sensitive to the effects of ROS. This dualeffect of sulindac was totally unexpected and surprising. Details areprovided in the examples which follow.

Polyps of the colon are precancerous growths that if left untreated canprogress to adenocarcinoma of the colon in a significant number ofpatients. Once a polyp is detected in the colon, surgical interventionis recommended to remove or destroy the lesion. However, the need existsto develop chemoprevention strategies for patients that cannot toleratepolypectomy or who are unwilling or unable to have a protectomy.Sulindac has been known as an anti-inflammatory and analgesic drug sincethe early 1970's. It is claimed in U.S. Pat. No. 3,654,349 issued toShen et al in 1972, and commercialized by Merck under the trade nameClinoril. Several NSAIDs, originally developed to treat arthritis, suchas sulindac have shown effectiveness in inhibiting and eliminatingcolonic polyps and in some cases, adenocarcinoma of the colon. Polypsvirtually disappear when the NSAID that the patient takes is sulindac.However, the prophylactic use of currently available NSAIDs, even inpolyposis syndrome patients, is marked by severe side reactions thatinclude gastrointestinal irritations and ulcerations. Once sulindactreatment is terminated due to such complications, the polyps canreturn, particularly in polyposis syndrome patients.

The compounds used in the treatment of this invention are effective onprecancerous and cancerous lesions either because they are activethemselves or because they are metabolized to active derivatives. In apreferred embodiment, a structurally related compound can be substitutedfor both compounds e.g. Sulindac and peroxides).

Oxidizing groups contemplated for use in accordance with the presentinvention include peroxides, nitrates, nitrites, perchlorates,chlorates, chlorites, hypochlorite, dichromates, permanganates, andpersulfates. Use of other electron accepting compounds including thosenot containing oxygen is within the scope of the invention as well.Bromine is an example of such a compound. Use of oxidizing agents orcompounds that cause cells to produce ROS such as arsenic trioxide,retinoic acid (and its derivatives), K antimonyl tartrate, doxorubicin,imexon, and bortezomib are within the scope of this invention.

Examples of peroxide compounds for utilization in the present inventioninclude, without limitation, hydrogen peroxide; organic peroxides;inorganic peroxides such as sodium peroxyborate tetrahydrate (sodiumperborate tetrahydrate) and calcium peroxide; peroxide complexes such asurea hydrogen peroxide, and superoxide salts such as sodium superoxide.In aqueous solution, the superoxide free radical (O₂*) dismutates (viareaction with its conjugate acid, the perhydroxyl free radical) to formhydrogen peroxide, and in biological systems the enzyme superoxidedismutase accelerates dismutation. Examples of organic peroxides includehydroperoxides (ROOH) such as lipid hydroperoxides, and internalperoxides such as artemisinin and its derivatives (an endoperoxide usedin the treatment of malaria). Elf Atochem, Inc. (Philadelphia, Pa.) is asource of many organic peroxide compounds. Hydrogen peroxide of highpurity can be obtained from Solvay Interox and other commercial sources.1-Hydroperoxycyclohexyl-1-hydroxy cyclohexyl peroxide and tert-Butylhydroperoxide can be obtained from Pfaltz and Bauer. Artemisinin can beobtained from Aldrich Chemical Company and Sigma Chemical Company.Sodium peroxyborate tetrahydrate can be obtained from Alfa JohnsonMatthey and from Fluka Chemika Biochemika. Urea hydrogen peroxide isavailable from Aldrich Chemical Company. Methods for the preparation offatty acid hydroperoxides (such as, for example, linolenic acidhydroperoxide, arachidonic acid hydroperoxide, and docosahexaenoic acidhydroperoxide) and of other lipid hydroperoxides (such as, for example,cholesterol hydroperoxide, cholesteryl linoleate hydroperoxide,trilinolein hydroperoxide, phosphatidylcholine hydroperoxide, andphosphatidylethanolamine hydroperoxide) are well known to those familiarwith the art. Many other peroxide compounds are available fromcommercial sources, and include 3-chloroperoxybenzoic acid,1,1-bis(tert-butylperoxy)cyclohexane, peracetic acid, monoperoxyphthalicacid, tert-butyl peroxide,2,5,bis(tert-butylperoxy)-2,5-dimethyl-3-hexyne, cumene hydroperoxide,2,5-bis(tert-butylperoxy)-2,5-dimethylhexane, lauroyl peroxide, benzoylperoxide, dicumyl peroxide, 2,5-dihydroperoxy-2,5-dimethylhexane,tert-butyl peracetate, tert-amyl hydroperoxide, diisononanoyl peroxide,decanoyl peroxide, succinic acid peroxide, 2,4-pentanedione peroxide,di-tert-butyl peroxide, tert-butyl cumyl peroxide,bis(t-butylperoxy)-diisopropylbenzene, and1-((hydroperoxycyclohexyl)dioxy)-cyclohexanol. As used in thisspecification, and in the claims appended hereto, the term peroxide orperoxide compound is meant to be inclusive of hydrogen peroxide,inorganic peroxides, organic peroxides, peroxide complexes, othercompounds containing the peroxy (peroxo) —O—O— moiety, superoxides, andperoxide precursor compounds which generate peroxide species in situ.Examples of organic peroxides include hydroperoxides, internalperoxides, endoperoxides, diacyl peroxides, ketone peroxides,peroxydicarbonates, peroxyesters, dialkyl peroxides, peroxyketals, andperoxyacids. Methods for the synthesis of organic peroxides are wellknown to those familiar with the art can be used in accordance with thepresent invention. Use of chromic acid, iodine, oxygen, ozone,peroxycarboxylic acids, permanganate, peroxyethanoic acid andperoxybenzoic acid is also contemplated. The primary effect of theoxidizing agent is to kill cellular material by binding a free radicalmolecule with the biological material and oxidizing the biologicalmaterial. Therefore, it is also believed that any biologically activefree radical, such as a heavy metal radical, may be effective.

Hydrogen peroxide has been used as a medicinal product for over acentury. The germicidal power of hydrogen peroxide is well known and isdue to the oxidizing effect of the peroxide. The main usage of hydrogenperoxide is in production of chemicals and bleaching of cellulose pulpand textiles. Small quantities are used for such purposes asdisinfection of eye contact lenses, disinfections of wounds and mouthwashing. Both hydrogen peroxide and carbamide peroxide are used for hairbleaching, oral antiseptics, dentifrices, oxidation of permanent waves,hair relaxers, ear drops, crank sores and tooth bleaching. Zinc peroxideis used as accelerator in rubber compounding, curing agent for syntheticelastomers and in cosmetic powders as antisepticals. Peroxide compoundsincluding hydrogen peroxide and carbamide peroxide have been used invarious dental procedures for many years. Reports of using peroxides tobleach or whiten teeth can be traced back to more than a century ago.Current peroxide containing whiteners used in USA can be classified into3 categories: 1) Those containing high concentration of hydrogenperoxide (30-35%) or carbamide peroxide (35%) for professional use only;2) materials that are dispensed by dentists and used by patients at home(up to 10% hydrogen peroxide or 16% carbamide peroxide); and 3)over-the-counter products with hydrogen peroxide content up to 6% andavailable to consumers for home use, such as Crest White Strips.

Hydrogen peroxide solutions of 35% or less would not be classified asskin irritants in rabbits by the European Union criteria (ECETOC, 1996).Skin irritation tests in rabbits with concentration of hydrogen peroxideof 3-8% were nonirritating to intact and abraded skin following exposurefor 24 hours under occlusive dressing (ECETOC, 1996). Irritation wasslight following 4 hour exposure to 10% hydrogen peroxide and mild with35% hydrogen peroxide.

The concentration of the sulindac in the topical formulation can rangefrom about 1% to 50% by weight. In one preferred embodiment, thesulindac is present in a concentration of about 5% or about 10% byweight. The carrier may further include humectants, fragrances, colors,thickeners, lubricants and preservatives, as is well known in the art.One particularly preferred humectant comprises a collagen derivedmaterial such as collagen laurate or the like. In some instances, thecarrier may simply comprise water, whereas in other instances it may bea lotion based carrier and may typically include ingredients such asglycerine, propylene glycol, methyl and/or propyl paraben, hydroxyalkylcellulose and the like. In one preferred embodiment, the carrier maycomprise a hypo-allergenic, high lipid, cream based carrier.

Sulindac has been particularly well received among the NSAIDs forgastrointestinal polyp treatment. Sulindac is a sulfoxide compound thatitself is believed to be inactive as an anti-arthritic agent. Thesulfoxide is known to be converted by liver and other tissues to thecorresponding sulfide, which is acknowledged to be the active moiety asa prostaglandin inhibitor. Recently, this conversion has been shown tobe catalyzed by methionine sulfoxide reductase (MsrA). The sulfide,however, is associated with the side effects of conventional NSAIDs.Sulindac appears to be metabolized to sulindac sulfone by as yet unknownreactions. Sulindac sulfone is not an inhibitor of prostaglandinsynthesis but has apoptotic activity against a wide array of cancercells. The sulfone is currently being evaluated in Phase 2-3 clinicaltrials as therapy for multiple different types of cancers.

Hydrogen peroxide concentrations used in various methods according tothe present invention can range from about 1% to 50% by weight and canbe varied according to exposure time. A preferred range is about 1%-20%.It is believed that cell death can occur with exposures toconcentrations of hydrogen peroxide as low as 3%. Preferably, theexposure time is less than 45 minutes, more preferably less than 15minutes, and most preferably no more than 10 minutes. If the oxidizingagent is delivered on a strip or in a gel or other matrix, the exposuretime may be very long and indefinite. One method utilizes aperoxide/adhesive mixture to provide a longer exposure.

When administered as a combination, the therapeutic agents can beformulated as separate compositions which are given at the same time ordifferent times, or the therapeutic agents can be given as a singlecomposition. The two components may be applied sequentially. In such asequential application, the sulindac will have to be applied firstfollowed by the peroxide. Preferably, the applications are made withinone hour and most preferably, they are made within about one half hourof each other.

Accordingly, the present invention may be implemented in connection witha treatment kit. The kit includes a composition having the sulindac andthe peroxide in a slow-release, two component delivery system such aswithin patches or strips at their effective concentrations, typicallyranging from about 5% to 6%. The kit will also preferably includeeducational materials for optimum patient compliance and follow-up.

Separate kits may be provided for use by physicians and patients. Thephysician's kit will include relatively higher concentrations of thematerials. It may be discovered that the effective concentrations forremoving a precancerous growth are different than the concentrationsneeded to destroy a BCC or SCC.

In a preferred embodiment, the methods and pharmaceutical compositionsof the present invention are used for the treatment or prevention ofneoplasia disorders including the group consisting of acral lentiginousmelanoma, actinic keratoses, adenocarcinoma, adenoid cystic carcinoma,adenomas, adenosarcoma, adenosquamous carcinoma, astrocytic tumors,bartholin gland carcinoma, basal cell carcinoma, bronchial glandcarcinomas, capillary, carcinoids, carcinoma, carcinosarcoma, cavernous,cholangiocarcinoma, chondosarcoma, choriod plexus papilloma/carcinoma,clear cell carcinoma, cystadenoma, endodermal sinus tumor, endometrialhyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma,ependymal, epithelioid, Ewing's sarcoma, fibrolamellar, focal nodularhyperplasia, gastrinoma, germ cell tumors, glioblastoma, glucagonoma,hemangioblastomas, hemangioendothelioma, hemangiomas, hepatic adenoma,hepatic adenomatosis, hepatocellular carcinoma, insulinoma,intraepithelial neoplasia, interepithelial squamous cell neoplasia,invasive squamous cell carcinoma, large cell carcinoma, leiomyosarcoma,lentigo maligna melanomas, leukoplakias, malignant melanoma, malignantmesothelial tumors, medulloblastoma, medulloepithelioma, melanoma,meningeal, mesothelial, metastatic carcinoma, mucoepidermoid carcinoma,neuroblastoma, neuroepithelial adenocarcinoma nodular melanoma, oat cellcarcinoma, oligodendroglial, osteosarcoma, pancreatic polypeptide,papillary serous adenocarcinoma, pineal cell, pituitary tumors,plasmacytoma, pseudosarcoma, pulmonary blastoma, renal cell carcinoma,retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, small cellcarcinoma, soft tissue carcinomas, somatostatin-secreting tumor,squamous carcinoma, squamous cell carcinoma, submesothelial, superficialspreading melanoma, undifferentiated carcinoma, uveal melanoma,verrucous carcinoma, vipoma, well differentiated carcinoma, and Wilm'stumor.

In another preferred embodiment, the pharmaceutical compositions of theinvention selectively kill all cancers or hyper-proliferativepre-malignant cells. Included in these cell types are cancers arisingfrom viruses or physical agents that would lead to hyper-proliferativestates. For example, pox viruses create a hyper-proliferative statesimilar to a cancer. HPV causes papillomas and cervical polyps(fibroids) that are not malignant but are proliferations that may beaffected by the combination.

In a preferred embodiment, a pharmaceutical composition comprisessulindac, sulindac metabolites, sulindac derivatives and an oxidatingagent. Preferably, the sulindac metabolite comprises sulindac sulfide orsulindac sulfone.

In another preferred embodiment, the sulindac comprises R- and S-epimersof sulindac. Preferably, the sulindac is an R-epimer. We have discoveredthat the R-epimer of sulindac can retain its cancer killing effects. Wehave also found that in normal cells, but not cancer cells, the R-epimerof sulindac is not efficiently converted to the sulfide, the active COXinhibitor. Since COX inhibitors produce serious toxicity, it is expectedthat the R-epimer of sulindac can be a potentially superior therapeuticagent because it would have a lower toxicity profile in normal cells.

Sulindac, unlike its metabolites, is a chiral compound since it containsa methyl sulfoxide moiety, in which there is asymmetry around the sulfuratom (FIG. 21). Therefore, the drug sulindac consists of an equalmixture of the R- and S-epimers, and would be a substrate for enzymesthat are specific for either epimer. Reduction of either epimer ofsulindac, which is a prodrug, will yield sulindac sulfide, which is theactive COX inhibitor. The R-epimer of sulindac would be advantageous toother compounds as it would not be efficiently converted to a COXinhibitor in normal cells in vivo.

In another preferred embodiment, the oxidating agent comprises at leastone of: peroxides, nitrates, nitrites, perchlorates, chlorates,chlorites, hypochlorite, dichromates, permanganates, and persulfates,bromine, arsenic trioxide, retinoic acid (and its derivatives), Kantimonyl tartrate, doxorubicin, imexon, and bortezomib, hydrogenperoxide; inorganic peroxides, sodium peroxyborate tetrahydrate (sodiumperborate tetrahydrate) calcium peroxide; peroxide complexes, ureahydrogen peroxide; superoxide salts, sodium superoxide; superoxide freeradical (O₂*) dismutates; organic peroxides, hydroperoxides (ROOH),lipid hydroperoxides, artemisinin and derivatives thereof;1-Hydroperoxycyclohexyl-1-hydroxy cyclohexyl peroxide, tert-Butylhydroperoxide; fatty acid hydroperoxides, linolenic acid hydroperoxide,arachidonic acid hydroperoxide, docosahexaenoic acid hydroperoxide,cholesterol hydroperoxide, cholesteryl linoleate hydroperoxide,trilinolein hydroperoxide, phosphatidylcholine hydroperoxide,phosphatidylethanolamine hydroperoxide; 3-chloroperoxybenzoic acid,1,1-bis(tert-butylperoxy)cyclohexane, peracetic acid, monoperoxyphthalicacid, tert-butyl peroxide,2,5,bis(tert-butylperoxy)-2,5-dimethyl-3-hexyne, cumene hydroperoxide,2,5-bis(tert-butylperoxy)-2,5-dimethylhexane, lauroyl peroxide, benzoylperoxide, dicumyl peroxide, 2,5-dihydroperoxy-2,5-dimethylhexane,tert-butyl peracetate, tert-amyl hydroperoxide, diisononanoyl peroxide,decanoyl peroxide, succinic acid peroxide, 2,4-pentanedione peroxide,di-tert-butyl peroxide, tert-butyl cumyl peroxide,bis(t-butylperoxy)-diisopropylbenzene, and1-((hydroperoxycyclohexyl)dioxy)-cyclohexanol; hydrogen peroxide,compounds containing a peroxy (peroxo) —O—O— moiety, superoxides,peroxide precursor compounds; endoperoxides, diacyl peroxides, ketoneperoxides, peroxydicarbonates, peroxyesters, dialkyl peroxides,peroxyketals, and peroxyacids. Preferably, the oxidating agent ishydrogen peroxide, and/or tert-butyl hydroperoxide (TBHP), and/orarsenic trioxide.

In another preferred embodiment, the oxidizing agent is hydrogenperoxide in a concentration is in a range of about 1% to 50% by weight.

In another preferred embodiment, the sulindac concentration is in arange from about 1% to 50% by weight.

In yet another embodiment, the oxidizing agent is arsenic trioxide in aconcentration in a range of about 1% to 50% by weight.

In another preferred embodiment, a topical pharmaceutical compositioncomprises a 5% sulindac gel and an oxidizing agent. Preferably, the 5%sulindac gel comprises: 71.08% deionized water; 12.00% SD Alcohol 40;10% KOH; 5% Sulindac; 1% Hydroxy methyl cellulose; 0.50% Xantham gum;0.20% Glydant Plus; 0.20% Citric Acid; and 0.02% disodium EDTA.

In other preferred embodiments, the sulindac gel is 10% sulindac, 15%sulindac, 20% sulindac, 50% sulindac.

In another preferred embodiment, a method of treating an abnormal cell,said method comprising administering to an abnormal cell apharmaceutical composition comprising sulindac, or sulindac metabolites,or sulindac derivatives or combinations thereof, and an oxidating agent,thereby treating an abnormal cell. Preferably, the sulindac metabolitecomprises sulindac sulfide or sulindac sulfone.

In another preferred embodiment, the oxidating agent comprises at leastone of: peroxides, nitrates, nitrites, perchlorates, chlorates,chlorites, hypochlorite, dichromates, permanganates, and persulfates,bromine, arsenic trioxide, retinoic acid (and its derivatives), Kantimonyl tartrate, doxorubicin, imexon, and bortezomib, hydrogenperoxide; inorganic peroxides, sodium peroxyborate tetrahydrate (sodiumperborate tetrahydrate) calcium peroxide; peroxide complexes, ureahydrogen peroxide; superoxide salts, sodium superoxide; superoxide freeradical (O₂*) dismutates; organic peroxides, hydroperoxides (ROOH),lipid hydroperoxides, artemisinin and derivatives thereof;1-Hydroperoxycyclohexyl-1-hydroxy cyclohexyl peroxide, tert-Butylhydroperoxide; fatty acid hydroperoxides, linolenic acid hydroperoxide,arachidonic acid hydroperoxide, docosahexaenoic acid hydroperoxide,cholesterol hydroperoxide, cholesteryl linoleate hydroperoxide,trilinolein hydroperoxide, phosphatidylcholine hydroperoxide,phosphatidylethanolamine hydroperoxide; 3-chloroperoxybenzoic acid,1,1-bis(tert-butylperoxy)cyclohexane, peracetic acid, monoperoxyphthalicacid, tert-butyl peroxide,2,5,bis(tert-butylperoxy)-2,5-dimethyl-3-hexyne, cumene hydroperoxide,2,5-bis(tert-butylperoxy)-2,5-dimethylhexane, lauroyl peroxide, benzoylperoxide, dicumyl peroxide, 2,5-dihydroperoxy-2,5-dimethylhexane,tert-butyl peracetate, tert-amyl hydroperoxide, diisononanoyl peroxide,decanoyl peroxide, succinic acid peroxide, 2,4-pentanedione peroxide,di-tert-butyl peroxide, tert-butyl cumyl peroxide,bis(t-butylperoxy)-diisopropylbenzene, and1-((hydroperoxycyclohexyl)dioxy)-cyclohexanol; hydrogen peroxide,compounds containing a peroxy (peroxo) —O—O— moiety, superoxides,peroxide precursor compounds; endoperoxides, diacyl peroxides, ketoneperoxides, peroxydicarbonates, peroxyesters, dialkyl peroxides,peroxyketals, and peroxyacids. Preferably, the oxidating agent ishydrogen peroxide and/or tert-butyl hydroperoxide (TBHP), and/or arsenictrioxide.

In another preferred embodiment, the oxidizing agent, hydrogen peroxide,concentration is in a range of about 1% to 50% by weight.

In another preferred embodiment, the sulindac concentration is in rangefrom about 1% to 50% by weight.

In another preferred embodiment, the oxidizing agent, arsenic trioxide,concentration is in a range of about 1% to 50% by weight.

In another preferred embodiment, the pharmaceutical compositioncomprises a 5% sulindac gel and an oxidizing agent. Preferably, the 5%sulindac gel comprises: 71.08% deionized water; 12.00% SD Alcohol 40;10% KOH; 5% Sulindac; 1% Hydroxy methyl cellulose; 0.50% Xantham gum;0.20% Glydant Plus; 0.20% Citric Acid; and 0.02% disodium EDTA.

In other preferred embodiments, the sulindac gel is 10% sulindac, 15%sulindac, 20% sulindac, 50% sulindac.

In yet another embodiment, a method of treating cancer, said methodcomprising administering to a cancer cell a pharmaceutical compositioncomprising sulindac, or sulindac metabolites, or sulindac derivatives orcombinations thereof, and an oxidating agent, thereby treating cancer.Sulindac, sulindac metabolites, sulindac derivatives and an oxidatingagent. Preferably, the sulindac metabolite comprises sulindac sulfide orsulindac sulfone.

In another preferred embodiment, the oxidating agent comprises at leastone of: peroxides, nitrates, nitrites, perchlorates, chlorates,chlorites, hypochlorite, dichromates, permanganates, and persulfates,bromine, arsenic trioxide, retinoic acid (and its derivatives), Kantimonyl tartrate, doxorubicin, imexon, and bortezomib, hydrogenperoxide; inorganic peroxides, sodium peroxyborate tetrahydrate (sodiumperborate tetrahydrate) calcium peroxide; peroxide complexes, ureahydrogen peroxide; superoxide salts, sodium superoxide; superoxide freeradical (O₂*) dismutates; organic peroxides, hydroperoxides (ROOH),lipid hydroperoxides, artemisinin and derivatives thereof;1-Hydroperoxycyclohexyl-1-hydroxy cyclohexyl peroxide, tert-Butylhydroperoxide; fatty acid hydroperoxides, linolenic acid hydroperoxide,arachidonic acid hydroperoxide, docosahexaenoic acid hydroperoxide,cholesterol hydroperoxide, cholesteryl linoleate hydroperoxide,trilinolein hydroperoxide, phosphatidylcholine hydroperoxide,phosphatidylethanolamine hydroperoxide; 3-chloroperoxybenzoic acid,1,1-bis(tert-butylperoxy)cyclohexane, peracetic acid, monoperoxyphthalicacid, tert-butyl peroxide,2,5,bis(tert-butylperoxy)-2,5-dimethyl-3-hexyne, cumene hydroperoxide,2,5-bis(tert-butylperoxy)-2,5-dimethylhexane, lauroyl peroxide, benzoylperoxide, dicumyl peroxide, 2,5-dihydroperoxy-2,5-dimethylhexane,tert-butyl peracetate, tert-amyl hydroperoxide, diisononanoyl peroxide,decanoyl peroxide, succinic acid peroxide, 2,4-pentanedione peroxide,di-tert-butyl peroxide, tert-butyl cumyl peroxide,bis(t-butylperoxy)-diisopropylbenzene, and1-((hydroperoxycyclohexyl)dioxy)-cyclohexanol; hydrogen peroxide,compounds containing a peroxy (peroxo) —O—O— moiety, superoxides,peroxide precursor compounds; endoperoxides, diacyl peroxides, ketoneperoxides, peroxydicarbonates, peroxyesters, dialkyl peroxides,peroxyketals, and peroxyacids. Preferably, the oxidating agent ishydrogen peroxide, and/or tert-butyl hydroperoxide (TBHP), and/orarsenic trioxide.

In another preferred embodiment, the oxidizing agent is hydrogenperoxide in a concentration is in a range of about 1% to 50% by weight.

In another preferred embodiment, the sulindac concentration is in rangefrom about 1% to 50% by weight.

In yet another embodiment, the oxidizing agent is arsenic trioxide in aconcentration in a range of about 1% to 50% by weight.

In another preferred embodiment, a topical pharmaceutical compositioncomprises a 5% sulindac gel and an oxidizing agent. Preferably, the 5%sulindac gel comprises: 71.08% deionized water; 12.00% SD Alcohol 40;10% KOH; 5% Sulindac; 1% Hydroxy methyl cellulose; 0.50% Xantham gum;0.20% Glydant Plus; 0.20% Citric Acid; and 0.02% disodium EDTA.

In other preferred embodiments, the sulindac gel is 10% sulindac, 15%sulindac, 20% sulindac, 50% sulindac.

Cancer Therapy: In accordance with the invention tumor target cells areselectively targeted by the compositions. Tumors can be the result ofinfection by a tumor causing virus or other means.

In another preferred embodiment, abnormal or cancer cells are targetedby the compositions. For example, many malignancies are associated withthe presence of foreign DNA, e.g. Bcr-Abl, Bcl-2, HPV.

The invention in general provides a method for treating diseases, suchas cancer and diseases which are caused by infectious agents such asviruses, bacteria, intra- and extra-cellular parasites, insertionelements, fungal infections, etc., which may also cause expression ofgene products by a normally unexpressed gene, abnormal expression of anormally expressed gene or expression of an abnormal gene.

The methods of the invention are preferably employed for treatment orprophylaxis against diseases caused abnormal cell growth and byinfectious agents, particularly for treatment of infections as may occurin tissue such as lung, heart, liver, prostate, brain, testes, stomach,intestine, bowel, spinal cord, sinuses, urinary tract or ovaries of asubject.

In another preferred embodiment, the compositions of the invention canbe administered in conjunction with chemotherapy. These chemotherapeuticagents can be co-administered, precede, or administered after thecompositions. Non-limiting examples of chemotherapeutic agents include,but not limited to: cyclophosphamide (CTX, 25 mg/kg/day, p.o.), taxanes(paclitaxel or docetaxel), busulfan, cisplatin, cyclophosphamide,methotrexate, daunorubicin, doxorubicin, melphalan, cladribine,vincristine, vinblastine, and chlorambucil.

In another preferred embodiment, the pharmaceutical composition,inhibits the tumor cell growth in a subject, and the method comprisesadministering to the subject a pharmaceutical composition comprising atherapeutically effective amount of the composition. Inhibition of tumorcell growth refers to one or more of the following effects: (1)inhibition, to some extent, of tumor growth, including, (i) slowing downand (ii) complete growth arrest; (2) reduction in the number of tumorcells; (3) maintaining tumor size; (4) reduction in tumor size; (5)inhibition, including (i) reduction, (ii) slowing down or (iii) completeprevention, of tumor cell infiltration into peripheral organs; (6)inhibition, including (i) reduction, (ii) slowing down or (iii) completeprevention, of metastasis; (7) enhancement of anti-tumor immuneresponse, which may result in (i) maintaining tumor size, (ii) reducingtumor size, (iii) slowing the growth of a tumor, (iv) reducing, slowingor preventing invasion and/or (8) relief, to some extent, of theseverity or number of one or more symptoms associated with the disorder.

Combination Therapies: The therapeutic compositions of the presentinvention may be combined with any other methods generally employed inthe treatment of the particular tumor, disease or disorder that thepatient exhibits. So long as a particular therapeutic approach is notknown to be detrimental to the patient's condition in itself, and doesnot significantly counteract the instant composition treatment, itscombination with the present invention is contemplated.

Therapeutic agents can include, for example, chemotherapeutic agentssuch as, cyclophosphamide (CTX, 25 mg/kg/day, p.o.), taxanes (paclitaxelor docetaxel), busulfan, cisplatin, methotrexate, daunorubicin,doxorubicin, melphalan, cladribine, vincristine, vinblastine,chlorambucil, tamoxifen, taxol, etoposide (VP-16), adriamycin,5-fluorouracil (5FU), camptothecin, actinomycin-D, mitomycin C,cisplatin (CDDP), combretastatin(s) and derivatives and prodrugsthereof.

As will be understood by those of ordinary skill in the art, theappropriate doses of chemotherapeutic agents will be generally aroundthose already employed in clinical therapies wherein thechemotherapeutics are administered alone or in combination with otherchemotherapeutics. By way of example only, agents such as cisplatin, andother DNA alkylating may be used. Cisplatin has been widely used totreat cancer, with efficacious doses used in clinical applications of 20mg/m² for 5 days every three weeks for a total of three courses.Cisplatin is not absorbed orally and must therefore be delivered viainjection intravenously, subcutaneously, intratumorally orintraperitoneally.

Further useful agents include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for adriamycin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of polynucleotideprecursors may also be used. Particularly useful are agents that haveundergone extensive testing and are readily available. As such, agentssuch as 5-fluorouracil (5-FU) are preferentially used by neoplastictissue, making this agent particularly useful for targeting toneoplastic cells. Although quite toxic, 5-FU, is applicable in a widerange of carriers, including topical, however intravenous administrationwith doses ranging from 3 to 15 mg/kg/day being commonly used.

The skilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, chapter 33, in particular pages 624-652, for non-limitingexamples of other chemotherapeutic agents that can be used incombination therapies with the PWM-poly IC-PHA compositions. Somevariation in dosage will necessarily occur depending on the condition ofthe subject being treated. The physician responsible for administrationwill be able to determine the appropriate dose for the individualsubject.

In connection solid tumor treatment, the present invention may be usedin combination with classical approaches, such as surgery, radiotherapy,chemotherapy, and the like. The invention therefore provides combinedtherapies in which the therapeutic compositions are used simultaneouslywith, before, or after surgery or radiation treatment; or areadministered to patients with, before, or after conventionalchemotherapeutic, radiotherapeutic or other anti-angiogenic agents, ortargeted immunotoxins or coaguligands.

When one or more agents are used in combination with thesulindac-peroxide compositions there is no requirement for the combinedresults to be additive of the effects observed when each treatment isconducted separately. Although at least additive effects are generallydesirable, any increased anti-tumor effect above one of the singletherapies would be of benefit. Also, there is no particular requirementfor the combined treatment to exhibit synergistic effects, although thisis certainly possible and advantageous.

To practice combined anti-tumor therapy, one would simply administer toan animal the composition in combination with another anti-cancer agentin a manner effective to result in their combined anti-tumor actionswithin the animal. The agents would therefore be provided in amountseffective and for periods of time effective to result in their combinedpresence within the tumor vasculature and their combined actions in thetumor environment. To achieve this goal, the compositions and otheranti-cancer agents may be administered to the animal simultaneously,either in a single composition, or as two distinct compositions usingdifferent administration routes.

Alternatively, the composition mediated treatment may precede, orfollow, the a second anti-cancer agent treatment by, e.g., intervalsranging from minutes to weeks. In certain embodiments where theanti-cancer agent and the composition are applied separately to theanimal, one would ensure that a significant period of time did notexpire between the time of each delivery, such that the anti-canceragent and the composition would still be able to exert an advantageouslycombined effect on the tumor. In such instances, it is contemplated thatone would contact the tumor with both agents within about 5 minutes toabout one week of each other and, more preferably, within about 12-72hours of each other, with a delay time of only about 12-48 hours beingmost preferred.

The general use of combinations of substances in cancer treatment iswell known. For example, U.S. Pat. No. 5,710,134 (incorporated herein byreference) discloses components that induce necrosis in tumors incombination with non-toxic substances or “prodrugs”. The enzymes setfree by necrotic processes cleave the non-toxic “prodrug” into the toxic“drug”, which leads to tumor cell death. Also, U.S. Pat. No. 5,747,469(incorporated herein by reference) discloses the combined use of viralvectors encoding p53 and DNA damaging agents. Any such similarapproaches can be used with the present invention.

In some situations, it may even be desirable to extend the time periodfor treatment significantly, where several days (2, 3, 4, 5, 6 or 7),several weeks (1, 2, 3, 4, 5, 6, 7 or 8) or even several months (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations. Thiswould be advantageous in circumstances where one treatment was intendedto substantially destroy the tumor, such as the sulindac-oxidecomposition treatment, and another treatment was intended to preventmicrometastasis or tumor re-growth, such as the administration of ananti-angiogenic agent.

It also is envisioned that more than one administration of either thecomposition or another anti-cancer agent will be utilized. Thecomposition and anti-cancer agents may be administered interchangeably,on alternate days or weeks; or a sequence of the composition treatmentmay be given, followed by a sequence of anti-cancer agent therapy. Inany event, to achieve tumor regression using a combined therapy, allthat is required is to deliver both agents in a combined amounteffective to exert an anti-tumor effect, irrespective of the times foradministration.

In terms of surgery, any surgical intervention may be practiced incombination with the present invention. In connection with radiotherapy,any mechanism for inducing DNA damage locally within tumor cells iscontemplated, such as γ-irradiation, X-rays, UV-irradiation, microwavesand even electronic emissions and the like. The directed delivery ofradioisotopes to tumor cells is also contemplated, and this may be usedin connection with a targeting antibody or other targeting means.

Cytokine therapy also has proven to be an effective partner for combinedtherapeutic regimens. Various cytokines may be employed in such combinedapproaches. Examples of cytokines include IL-1α, IL-10, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, TGF-β,GM-CSF, M-CSF, G-CSF, TNFα, TNFβ, LAF, TCGF, BCGF, TRF, BAF, BDG, MP,LIF, OSM, TMF, PDGF, IFN-α, IFN-β, IFN-γ. Cytokines are administeredaccording to standard regimens, consistent with clinical indicationssuch as the condition of the patient and relative toxicity of thecytokine. Uteroglobins may also be used to prevent or inhibit metastases(U.S. Pat. No. 5,696,092; incorporated herein by reference).

Effective Amounts

The compositions described above are preferably administered to asubject in an effective amount. An effective amount is an amount whichis capable of producing a desirable result in a treated animal or cell(for example, to induce apoptosis or impair mitosis in a cell in theanimal or a culture). As is well known in the medical and veterinaryarts, dosage for any one animal depends on many factors, including theparticular animal's size, body surface area, age, the particularcomposition to be administered, time and route of administration,general health, and other drugs being administered concurrently. It isexpected that an appropriate dosage for topical administration of thesulindac in the topical formulation can range from 1% to 25%. In thepreferred embodiment, the sulindac is present in a concentration of 5%or 10%. An effective amount for use with a cell in culture will alsovary, but can be readily determined empirically (for example, by addingvarying concentrations to the cell and selecting the concentration thatbest produces the desired result). It is expected that an appropriateconcentration would be in the range of about 1-5000 μM.

Method for Inhibiting Cancer Cell Growth

The invention provides a method for inhibiting tumor cell growth orincreasing the rate of tumor cell apoptosis. The method includes thesteps of contacting a tumor cell with a composition including asufficient amount of sulindac-oxide compositions to kill or at leastretard mitosis in the tumor cell. The method may be used to inhibit thegrowth of numerous types of cancerous tumor cells. The compositions havebeen tested and shown to be effective against different types of tumorssuch as melanoma, squamous, and breast cancer cells. (See, exampleswhich follow). Sulindac (or related compounds thereof) comprisingcompositions are expected to be effective against other cancers as well,particularly those derived from epithelial, mesenchymal, and hemopoieticorigins.

Any suitable formulation of sulindac-peroxide can be used in methods ofthe invention. Typical formulations are topical liposomal formulationsof the compositions of varying concentrations. In addition to topicaladministration, sulindac-peroxide containing formulations can beadministered to a subject via injection (e.g., IP, IV, IM, SQ).

In preferred embodiments, administration of sulindac-oxide compositionsresults in one or more phenotypes of a tumor cell being inhibited. Forexample, inhibition of tumor growth, reduction of tumor size, inhibitionof metastasis, reduction in the number of tumor cells and the like. Eachof these phenotypes of a tumor cell can be measured using standardassays, such as for example, imaging, mechanical measurements, in vitroassays and the like.

Formulations

A compound of the present invention can be formulated as apharmaceutical composition. Such a composition can then be administeredorally, parenterally, by inhalation spray, rectally, or topically indosage unit formulations containing conventional nontoxicpharmaceutically acceptable carriers, adjuvants, and vehicles asdesired. Topical administration can also involve the use of transdermaladministration such as transdermal patches or iontophoresis devices. Theterm parenteral as used herein includes subcutaneous injections,intravenous, intramuscular, intrasternal injection, inhalation orinfusion techniques.

Suppositories for rectal administration of the drug can be prepared bymixing the drug with a suitable nonirritating excipient such as cocoabutter, synthetic mono- di- or triglycerides, fatty acids andpolyethylene glycols that are sold at ordinary temperatures but liquidat the rectal temperature and will therefore melt in the rectum andrelease the drug.

The methods and combinations of the present invention provide one ormore benefits. Combinations of the present invention may allow for alower dose of each agent. A benefit of lowering the dose of thecompounds, compositions, agents and therapies of the present inventionadministered to a mammal includes a decrease in the incidence of adverseeffects associated with higher dosages.

By lowering the incidence of adverse effects, an improvement in thequality of life of a patient undergoing treatment for cancer iscontemplated. Further benefits of lowering the incidence of adverseeffects include an improvement in patient compliance, a reduction in thenumber of clinical visits needed for the treatment of adverse effects,and a reduction in the administration of analgesic agents needed totreat pain associated with the adverse effects.

Alternatively, the methods and combination of the present invention canalso maximize the therapeutic effect at higher doses.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of where treatment is required, such as liniments, lotions,creams, ointments or pastes, and drops suitable for administration tothe eye, ear, or nose. Drops according to the present invention maycomprise sterile aqueous or oily solutions or suspensions and may beprepared by dissolving the active ingredient in a suitable aqueoussolution of a bactericidal and/or fungicidal agent and/or any othersuitable preservative, and preferably including a surface active agent.The resulting solution may then be clarified and sterilized byfiltration and transferred to the container by an aseptic technique.Examples of bactericidal and fungicidal agents suitable for inclusion inthe drops are phenylmercuric nitrate or acetate (0.002%), benzalkoniumchloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solventsfor the preparation of an oily solution include glycerol, dilutedalcohol and propylene glycol.

The composition of the invention can be administered to a patient eitherby themselves, or in pharmaceutical compositions where it is mixed withsuitable carriers or excipient(s). In treating a patient exhibiting adisorder of interest, a therapeutically effective amount of a agent oragents such as these is administered. A therapeutically effective doserefers to that amount of the compound that results in amelioration ofsymptoms or a prolongation of survival in a patient.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Thedata obtained from these cell culture assays and animal studies can beused in formulating a range of dosage for use in human. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating plasma concentration range that includes theIC₅₀ as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by HPLC.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (Seee.g. Fingl et al., in The Pharmacological Basis of Therapeutics, 1975,Ch. 1 p. 1). It should be noted that the attending physician would knowhow to and when to terminate, interrupt, or adjust administration due totoxicity, or to organ dysfunctions. Conversely, the attending physicianwould also know to adjust treatment to higher levels if the clinicalresponse were not adequate (precluding toxicity). The magnitude of anadministrated dose in the management of the oncogenic disorder ofinterest will vary with the severity of the condition to be treated andto the route of administration. The severity of the condition may, forexample, be evaluated, in part, by standard prognostic evaluationmethods. Further, the dose and perhaps dose frequency, will also varyaccording to the age, body weight, and response of the individualpatient. A program comparable to that discussed above may be used inveterinary medicine.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in Remington'sPharmaceutical Sciences, 18^(th) ed., Mack Publishing Co., Easton, Pa.(1990). Suitable routes may include oral, rectal, transdermal, vaginal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections, just to name afew.

The compositions described above may be administered to a subject in anysuitable formulation. In addition to treatment of cancer with topicalformulations of the composition, in other aspects of the invention thecomposition can be delivered by other methods. For example, thecomposition can be formulated for parenteral delivery, e.g., forsubcutaneous, intravenous, intramuscular, or intratumoral injection.Other methods of delivery, for example, liposomal delivery or diffusionfrom a device impregnated with the composition might be used. Thecompositions may be administered in a single bolus, multiple injections,or by continuous infusion (for example, intravenously or by peritonealdialysis). For parenteral administration, the compositions arepreferably formulated in a sterilized pyrogen-free form. Compositions ofthe invention can also be administered in vitro to a cell (for example,to induce apoptosis in a cancer cell in an in vitro culture) by simplyadding the composition to the fluid in which the cell is contained.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in Remington'sPharmaceutical Sciences, 18^(th) ed., Mack Publishing Co., Easton, Pa.(1990). Suitable routes may include oral, rectal, transdermal, vaginal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections, just to name afew.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Forsuch transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

Use of pharmaceutically acceptable carriers to formulate the compoundsherein disclosed for the practice of the invention into dosages suitablefor systemic administration is within the scope of the invention. Withproper choice of carrier and suitable manufacturing practice, thecompositions of the present invention, in particular, those formulatedas solutions, may be administered parenterally, such as by intravenousinjection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient to be treated.

Agents intended to be administered intracellularly may be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents may be encapsulated into liposomes, thenadministered as described above. Liposomes are spherical lipid bilayerswith aqueous interiors. All molecules present in an aqueous solution atthe time of liposome formation are incorporated into the aqueousinterior. The liposomal contents are both protected from the externalmicroenvironment and, because liposomes fuse with cell membranes, areefficiently delivered into the cell cytoplasm. Additionally, due totheir hydrophobicity, small organic molecules may be directlyadministered intracellularly.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein. Inaddition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions. The pharmaceuticalcompositions of the present invention may be manufactured in a mannerthat is itself known, e.g., by means of conventional mixing, dissolving,granulating, dragee-making, levitating, emulsifying, encapsulating,entrapping or lyophilizing processes.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of where treatment is required, such as liniments, lotions,creams, ointments or pastes, and drops suitable for administration tothe eye, ear, or nose. Drops according to the present invention maycomprise sterile aqueous or oily solutions or suspensions and may beprepared by dissolving the active ingredient in a suitable aqueoussolution of a bactericidal and/or fungicidal agent and/or any othersuitable preservative, and preferably including a surface active agent.The resulting solution may then be clarified and sterilized byfiltration and transferred to the container by an aseptic technique.Examples of bactericidal and fungicidal agents suitable for inclusion inthe drops are phenylmercuric nitrate or acetate (0.002%), benzalkoniumchloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solventsfor the preparation of an oily solution include glycerol, dilutedalcohol and propylene glycol.

Lotions according to the present invention include those suitable forapplication to the skin or eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those for the preparation of drops. Lotions orliniments for application to the skin may also include an agent tohasten drying and to cool the skin, such as an alcohol or acetone,and/or a moisturizer such as glycerol or an oil such as castor oil orarachis oil.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient infinely-divided or powdered form, alone or in solution or suspension inan aqueous or non-aqueous fluid, with the aid of suitable machinery,with a greasy or non-greasy basis. The basis may comprise hydrocarbonssuch as hard, soft or liquid paraffin, glycerol, beeswax, a metallicsoap; a mucilage; an oil of natural origin such as almond, corn,arachis, castor or olive oil; wool fat or its derivatives, or a fattyacid such as stearic or oleic acid together with an alcohol such aspropylene glycol or macrogels. The formulation may incorporate anysuitable surface active agent such as an anionic, cationic or non-ionicsurface active such as sorbitan esters or polyoxyethylene derivativesthereof. Suspending agents such as natural gums, cellulose derivativesor inorganic materials such as silicaceous silicas, and otheringredients such as lanolin, may also be included.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxy-methylcellulose, and/orpolyvinyl pyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coating. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

The composition can include a buffer system, if desired. Buffer systemsare chosen to maintain or buffer the pH of compositions within a desiredrange. The term “buffer system” or “buffer” as used herein refers to asolute agent or agents which, when in a water solution, stabilize suchsolution against a major change in pH (or hydrogen ion concentration oractivity) when acids or bases are added thereto. Solute agent or agentswhich are thus responsible for a resistance or change in pH from astarting buffered pH value in the range indicated above are well known.While there are countless suitable buffers, potassium phosphatemonohydrate is a preferred buffer.

The final pH value of the pharmaceutical composition may vary within thephysiological compatible range. Necessarily, the final pH value is onenot irritating to human skin and preferably such that transdermaltransport of the active compound, i.e. sulindac, peroxide, arsenictrioxide is facilitated. Without violating this constraint, the pH maybe selected to improve the compound stability and to adjust consistencywhen required. In one embodiment, the preferred pH value is about 3.0 toabout 7.4, more preferably about 3.0 to about 6.5, most preferably fromabout 3.5 to about 6.0.

For preferred topical delivery vehicles the remaining component of thecomposition is water, which is necessarily purified, e.g., deionizedwater. Such delivery vehicle compositions contain water in the range ofmore than about 50 to about 95 percent, based on the total weight of thecomposition. The specific amount of water present is not critical,however, being adjustable to obtain the desired viscosity (usually about50 cps to about 10,000 cps) and/or concentration of the othercomponents. The topical delivery vehicle preferably has a viscosity ofat least about 30 centipoises.

Other known transdermal skin penetration enhancers can also be used tofacilitate delivery of the composition. Illustrative are sulfoxides suchas dimethylsulfoxide (DMSO) and the like; cyclic amides such as1-dodecylazacycloheptane-2-one (Azone™, a registered trademark of NelsonResearch, Inc.) and the like; amides such as N,N-dimethyl acetamide(DMA) N,N-diethyl toluamide, N,N-dimethyl formamide, N,N-dimethyloctamide, N,N-dimethyl decamide, and the like; pyrrolidone derivativessuch as N-methyl-2-pyrrolidone, 2-pyrrolidone,2-pyrrolidone-5-carboxylic acid, N-(2-hydroxyethyl)-2-pyrrolidone orfatty acid esters thereof, 1-lauryl-4-methoxycarbonyl-2-pyrrolidone,N-tallowalkylpyrrolidones, and the like; polyols such as propyleneglycol, ethylene glycol, polyethylene glycol, dipropylene glycol,glycerol, hexanetriol, and the like; linear and branched fatty acidssuch as oleic, linoleic, lauric, valeric, heptanoic, caproic, myristic,isovaleric, neopentanoic, trimethyl hexanoic, isostearic, and the like;alcohols such as ethanol, propanol, butanol, octanol, oleyl, stearyl,linoleyl, and the like; anionic surfactants such as sodium laurate,sodium lauryl sulfate, and the like; cationic surfactants such asbenzalkonium chloride, dodecyltrimethylammonium chloride,cetyltrimethylammonium bromide, and the like; non-ionic surfactants suchas the propoxylated polyoxyethylene ethers, e.g., Poloxamer 231,Poloxamer 182, Poloxamer 184, and the like, the ethoxylated fatty acids,e.g., Tween 20, Myrj 45, and the like, the sorbitan derivatives, e.g.,Tween 40, Tween 60, Tween 80, Span 60, and the like, the ethoxylatedalcohols, e.g., polyoxyethylene (4) lauryl ether (Brij 30),polyoxyethylene (2) oleyl ether (Brij 93), and the like, lecithin andlecithin derivatives, and the like; the terpenes such as D-limonene,α-pinene, β-carene, α-terpineol, carvol, carvone, menthone, limoneneoxide, α-pinene oxide, eucalyptus oil, and the like. Also suitable asskin penetration enhancers are organic acids and esters such assalicyclic acid, methyl salicylate, citric acid, succinic acid, and thelike.

Kits and Formulations

The invention also provides a kit for reducing the rate of tumor growthin a subject. The kit of the invention includes a composition comprisingsulindac, sulindac derivatives, variants etc, oxides such as hydrogenperoxide, arsenic trioxide etc, and, optionally a pharmaceuticallyacceptable carrier as well as printed instructions for using thecomposition to reduce the rate of tumor growth in a subject.

Active components can be present in solid, semi-solid or liquid form.Solid forms include for example, powders, granules and flakes.Semi-solid forms include, for example, gels, creams, gelatins andointments. These and other active agents embraced by the presentinvention are known to those of ordinary skill in the art and, in mostcases, are commercially available from suppliers such as CompoundSolutions, Inc., Escondido, Calif. Information on these and other activeand inactive agents embraced by the invention, and their commercialsuppliers is available from various trade manuals, most particularly,Remington's Pharmaceutical Sciences, United States Pharmacopoeia (USP),National Formulary (NF), Merck Index, Physician's Desk Reference (PDR)and Chemical Abstracts.

The kits of the invention will also generally contain at least oneinactive agent. As used herein, inactive agents are agents which do notprovide any therapeutic benefit to the subject to whom they areadministered. Instead, inactive agents can function in many other wayssuch as to provide a base in which the active agent can be dissolved orsuspended, to dilute the active agent in order to provide proper dosesupon administration, to facilitate the dissolution or suspension of theactive agent, or to prevent oxidation of the active agent by removingair bubbles from the final compounded suspension. In some embodiments ofthe invention, the kits lack an inactive agent, and rather contain twoor more active agents.

Base agents such as creams, oils, gels or ointments are suitable fortopical or suppository applications. The choice of suitable inactivebase agent for use in the kits of the invention will depend upon theactive agent to be compounded. Suitable base agents will be known to theordinary artisan. Alternatively, Remington's Pharmaceutical Sciences,the Physician Desk Reference (PDR) or other manuals as listed above, canbe consulted in making this determination.

Examples of inactive base agents or components include, for example,lanolin, hydrophilic ointment, white ointment, yellow ointment,polyethylene glycol ointment, petrolatum, hydrophilic petrolatum, whitepetrolatum, rose water ointment, squalene, hydrogenated vegetable oil(Type II), ultrasound gel, pluronic lecithin organogel (PLO) gel, cream.

The term “petrolatum” as used herein means petrolatum ointment,petrolatum gel or petrolatum cream, all of which are commerciallyavailable. It is well within the realm of the ordinary pharmaceuticalartisan to determine which form of petrolatum is most appropriate for aspecific kit.

A commercially available ultrasound base is either POLYSONIC™(ultrasound gel) ultrasound lotion or Aquasonic ultrasound 100 gelmanufactured by Parker Laboratories, Inc. (Fairfield, N.J.) or EcoGel100 or EcoGel 200 manufactured by Eco-Med (Mississauga, Ontario,Canada), the compositions of which may include cetyl alcohol, liquidparaffin, polymer, surfactants, preservatives such as propyl paraben andmethyl paraben in bacteriostatic concentration, fragrance, and reverseosmosis water. As used herein, a gel is a base with a higher viscositythan a lotion. The physical characteristics of the POLYSONIC™(ultrasound gel) ultrasound lotion and the EcoGel 100 include pH rangeof 6.5-7.0, density of 1.04 g/cm³, viscosity of 35,000 to 70,000 cps andacoustic impedence of 1.60 (105 g/cm2 sec). The physical characteristicsof Aquasonic ultrasound 100 gel or EcoGel 200 are similar to those ofPOLYSONIC™ (ultrasound gel) ultrasound lotion and EcoGel 100 except thattheir viscosity is 80,000 to 110,000 cps. These lotions and gels areavailable in a clear, colorless form or in a blue colored form.

Liquid bases are recommended for orally administered pharmaceuticals. Insome embodiments of the invention, at least one active agent, will besupplied already co-mingled with an inactive agent. Examples of thisinclude the combination of magnesium hydroxide and aluminum hydroxide(commercially available as MAALOX™ (magnesium hydroxide/aluminumhydroxide)), and diphenhydramine HCl (commercially available asBENADRYL™ (diphenhydramine hydrochloride)). Both MAALOX™ (magnesiumhydroxide/aluminum hydroxide) and BENADRYL™ (diphenhydraminehydrochloride) are supplied by their respective manufacturers as acombination of active and inactive agents.

Sterile base solutions are preferred for parenteral (i.e., injection),aerosol (i.e., inhalation) and ophthalmic routes of administration. Theadministration may, for example, be intravenous, intraperitoneal,intramuscular, intracavity, subcutaneous or transdermal. Preparationsfor parenteral administration includes sterile aqueous or nonaqueoussolutions, suspensions and emulsions. The compounded pharmaceuticals,preferably those intended for parenteral, inhalation or ophthalmicroutes of administration, may be prepared and administered in inactiveagents which are pharmaceutically-acceptable. As used herein, apharmaceutically-acceptable carrier means a non-toxic material that doesnot interfere with the effectiveness of the biological activity of theactive agents and that is compatible with the biological systems such ofa tissue or organism. The physiologically acceptable carrier must besterile for in vivo administration. Pharmaceutically acceptable carriersinclude diluents, fillers, salts, buffers, stabilizers, solubilizers andother materials which are well-known in the art. The characteristics ofthe carrier will depend on the route of administration. In general,pharmaceutically-acceptable agents or carriers are well-known to thoseof ordinary skill in the art. In some embodiments, suitable sterilesolutions include albuterol and ipratropium inhalation solution;papaverine, phentolamine and prostaglandin injection solution; fentanylcitrate injection solution and cyclosporine ophthalmic drops.

Examples of nonaqueous solvents are propylene glycol, polyethyleneglycol, vegetable oil such as olive oil, an injectable organic esterssuch as ethyloliate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers, (such as those based on Ringer's dextrose),and the like. Preservatives and other additives may also be present suchas, for example, antimicrobials, antioxidants, chelating agents, andinert gases and the like. Those of skill in the art can readilydetermine the various parameters for preparing these alternativepharmaceutical compositions without resort to undue experimentation.

Inactive agents may also include components which function to preservethe integrity of the compounded formulation. This latter category ofinactive agents includes, for example, anti-foaming agents. Anti-foamingagents are agents which function to remove unwanted air trapped in acomposition, perhaps during mixing or agitation. The use of anti-foamingcomponents is particularly useful in the preparation of pharmaceuticalsto be used for ultrasound imaging due to the impedance of signaltransmission by air bubbles. Examples of other anti-foaming agentsuseful in the compositions of the invention includebisphenylhexamethicone, dimethicone, dimethiconol, hexamethyldisiloxane,hexyl alcohol, isopropyl alcohol, petroleum distillates, phenethyldisiloxane, phenyl trimethicone, polysilicone-7, propyl alcohol, silicadimethyl silylate, silica silylate, tetramethyl decynediol andtrimethylsiloxysilicate. A preferred anti-foaming agent is simethicone.Simethicone is a mixture of about 90% dimethicone and 10% siliconedioxide (w/w). Simethicone is used extensively as an anti-gas agent inpharmaceutical products such as GAS-X™ (simethicone), MAALOX™ (magnesiumhydroxide/aluminum hydroxide), MYLANTA™ (aluminum, magnesiumsimethicone), PHAZYME™ (simethicone), GENAZYME™ (simethicone), andMYLICON™ (simethicone) Drops. Simethicone may be used as an anti-foamingagent in any of the formulations embraced by the invention.

Other inactive agents which can be included in the formulations of theinvention include stabilizers such as citric acid, anti-oxidants such assodium metabisulfite and preservatives such as methyl or propyl paraben.

Another class of inactive agents is suspending agents. Suspending agentsare agents which facilitate the suspension and in some cases thedissolution of an active agent in a base. Generally, suspending agentsensure more uniform mixing of active and base components. In order toadminister a more uniform dose of a compounded pharmaceutical to apatient, the compounded components must be properly and homogeneouslycombined. If the active agent is present as a powder, a uniformdispersion is sometimes difficult to achieve using the traditional formof compounding.

A subcategory of suspending agents are solubilizers. Solubilizers areagents which facilitate the dissolution of a solid or, in some cases, asemi-solid agent in a base inactive agent. In some embodiments of theinvention, a solid-form active agent may be dissolved in a suspendingagent, prior to mixing it with the base agent. Conversely, thesuspending agent and the base agent may be prepackaged together,particularly if the concern is ensuring the uniform blending of activeagent within the base component rather than the loss of solid (i.e.,powdery) active agent. In still other variations, the suspending agentmay be premixed with the base inactive agent.

Suitable suspending agents useful in the compositions of the inventioninclude, but are not limited to, glycerin, hexylene glycol, propyleneglycol, sorbitol, acacia, cholesterol, diethanolamine (adjunct),glyceryl monostearate, lanolin alcohols, lecithin, mono- anddi-glycerides, monoethanolamine (adjunct), oleic acid (adjunct), oleylalcohol (stabilizer), poloxamer, polyoxyethylene 50 stearate, polyoxyl35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyl 10 oleylether, polyoxyl 20 cetostearyl ether, polyoxyl 40 stearate, polysorbate20, polysorbate 40, polysorbate 60, polysorbate 80, propylene glycoldiacetate, propylene glycol monostearate, sodium lauryl sulfate, sodiumstearate, sorbitan monolaurate, sorbitan monooleate, sorbitanmonopalmitate, sorbitan monostearate, stearic acid, trolamine,emulsifying wax, benzalkonium chloride, benzethonium chloride,cetylpyridinium chloride, docusate sodium, nonoxynol 9, nonoxynol 10,octoxynol 9, polyoxyl 50 stearate, and tyloxapol.

Still other suspending agents include humectants and wetting agents.Humectants are agents which retain moisture. Examples of humectantsinclude but are not limited to glycerin, hexylene glycol, propyleneglycol and sorbitol. The amounts of base and non-base inactive agentswill also depend upon the particular compounded pharmaceutical to bemade. Base agents can be provided in quantities corresponding to finalcompounded preparations which contain 0.5% to 99.99% of base agent,either in weight or in volume. In preferred embodiments, the finalconcentration of the base agent is 20%-80%. In even more preferredembodiments, the final concentration of the base agent is 40%-80%.

Generally, the amounts of non-base agents will be sufficient to providefinal formulations in which each non-base inactive agent represents0.01%-50% (w/w) of the composition. Suspending agents may represent1%-50% (w/w) of the final formulation. Preferably, suspending agentswill represent 1%-40% and even more preferably, they will represent5%-30% of the final formulation. Anti-foaming agents may represent 0.01%to 20% (w/w) of the final formulation. More preferably, anti-foamingagents represent 0.05% to 10% of the final formulation and even morepreferably, they represent 0.1% to 5% of the final formulation.

In some preferred embodiments, the single or multiple unit of use kitsare designed to yield, after the physical mixing of active and inactiveagents, compounded pharmaceutical formulations comprising 0.1%, 5%, 10%to 99% w/w of sulindac or variants thereof.

The kits of the invention will provide each and every component requiredfor preparing a given compounded pharmaceutical in pre-measuredquantities. The measuring of each component will be performed usingcurrent Good Manufacturing Practices (cGMP, as legislated by the Code ofFederal Regulations or CFR), as will the packaging and labeling of eachcomponent and the final packaging and labeling of the kit in itsentirety. In this way, the kits are standardized and variations frombatch to batch will be minimal or non-existent and the precision andaccuracy in the measurement of individual components will be improvedconsiderably over the methods currently used by pharmacists.Instructions may be provided as separate from any container, but stillcontained in the kit. Alternatively, instructions may be located on acontainer, for example, on an exterior surface or on an interior surfacesuch as a lid.

Both the active and the inactive agents of the kit are provided incontainers. Since the kit will contain at least one active and at leastone inactive agent, or at least two active agents pre-formulated withinactive agents, the minimum number of containers in a given kit will betwo. In preferred embodiments, the maximum number of containers in a kitwill be less than or equal to four. The containers may be formed in anysize or shape useful for the mixing or transferring of components fromone container to another. For example, each container may be in the formof vials, bottles, squeeze bottles, jars, sealed sleeves, envelopes orpouches, tubes or blister packages or any other suitable form providedthe container is sealed so as to prevent premature mixing of components.As used herein, a container may also be a compartment or a chamberwithin a vial, a tube, ajar, or an envelope, or a sleeve, or a blisterpackage or a bottle, provided that the contents of one compartment arenot able to associate physically with the contents of anothercompartment prior to their deliberate mixing by a pharmacist orphysician.

The invention intends to provide within a single kit all the necessarycomponents, containers and stirring or mixing elements for preparing aunit of use compounded pharmaceutical without the need for otheraccessories. The kits of the invention may also contain items such asgloves or spill pads. Individuals skilled in the art can readily modifythe choice of container to suit the individual components housed andmixed therein.

In some embodiments of the invention, the final compounded formulationwill be provided to the patient in the container originally housing theinactive, or base, compound. In other embodiments, the final compoundedformulation will be provided in the container originally housing theactive agent. In still other embodiments, all the necessary componentsfor preparing a compounded pharmaceutical are included in one containerbut are physically separated within such a container. For example, aninactive agent may be contained in the lower part of a container, suchas a jar, and may be covered by a plastic, peel-off wrap. The activeagent may be housed in this same jar, but secured to the lid of the jarand provided in a pouch or a sleeve. The ability to provide allcomponents together in the smallest packaging arrangement may bepreferable in some circumstances. Mixing elements required in thepreparation of the compounded pharmaceutical may also be located withinthe same container, for example, secured to the inside surface of thelid of the container.

In still another embodiment of the invention, active and inactive agentsare provided in adjacent compartments of a single housing container, andare mechanically removed from these compartments and into a thirdcompartment. As an example, all the chemical components necessary toprepare a particular compounded pharmaceutical can be present in asingle tube, for example, a tube similar to a toothpaste tube having aninterior which is divided into separate compartments. Each of thesecompartments in turn house a base agent or an active agent. Either thebase agent or the active agent may be premixed with an anti-foamingagent and/or a suspending agent, as described herein. By applyingpressure on the tube as a whole, the components are made to exit theirrespective compartments. They can then be mixed either in an adjacent ora physically separate compartment. Squeezing or pressing of the outsidesurface of the tube may be all that is necessary to retrieve theindividual components housed within the tube. In yet another embodiment,the contents of both chambers of a container can be pumped out and intoa third container. In a related embodiment, it is also envisioned thatrather than requiring the contents of each compartment to exit and flowinto a third compartment, the components may be separated by a removablesheet or film. Thus, upon removal of such a sheet or film, the contentsof the two compartments are in contact and may require only agitation orend-over-end inversion to become completely mixed. This latterembodiment would eliminate the need for a mixing element, andpotentially for an exterior package particularly if the instructions arewritten on the container itself.

According to some aspects of the invention, each container may containone or more active agents or one or more inactive agents. For example,in some embodiments of the invention, none of the containers may containboth an active and an inactive agent prior to mixing by the pharmacistor physician. However, the invention also provides for kits in which acontainer may contain an active and at least one inactive agent, such asa base agent, a suspending agent or an anti-foaming agent.

In a preferred embodiment, the active agent is provided premixed with aninactive agent. This applies mainly when sulindac will be commerciallyavailable as a solid, for example a powder, and the pre-mixing of thepowder with a suspending agent facilitates the compounding by thepharmacist or physician. In yet other embodiments, at least two of theinactive agents may be pre-mixed as provided in the kits of theinvention.

In some embodiments, where the active agent is added to the basecomponent, it may be desirable to provide the base component in acontainer which is only partially full. In preferred embodiments, thecontainer in which the base component is situated is less than 100% fullby volume. In other embodiments, the containers are 95%, 90%, 80%, 75%,70%, 60%, 50%, 40%, 30%, 25%, 20% or less than 20% full by volume. Inother embodiments, the active or inactive agents comprise a volume oftheir respective containers ranging from 100% to greater than 1%, andevery integer there between. In preferred embodiments, the inactiveagent occupies a volume of the second container which is less than orequal to the volume of the second container minus the volume of theactive agent.

As used according to the invention, the active and inactive agents arephysically combined by a pharmacist to produce a compoundedpharmaceutical. The components of the kit can be combined by gentleagitation, shaking, stirring, folding or end-over-end inversion of thefirst or second container. In some instances, the proper mixing of theactive and inactive agents may be accomplished simply by adding one tothe other, followed by sealing and agitation of the container. This isespecially the case if the components are both liquids or bothsemi-solids. In other instances, it may be necessary to stir thecomponents together with a mixing element. Mixing elements are wellknown to a person of ordinary skill in the pharmaceutical arts and mayinclude for example, centrifuges, a mixing rod such as a glass rod, aspoon, a spatula or a dipstick. Where required, the mixing element isprovided in the kit. The presence of a mixing element will varydepending on the compounded pharmaceutical formulation to be made withthe components of a kit.

The final compounded pharmaceutical may be formulated into preparationsin solid, semi-solid, liquid or gaseous forms such as tablets, capsules,powders, granules, ointments, solutions, suppositories, inhalants andinjections, and usual ways for oral, parenteral or surgicaladministration. The invention also embraces locally administering thecompounded pharmaceuticals of the invention such as, for example, asimplants. These formulations may be intended for oral, topical, mucosal,parenteral (e.g., injectable), rectal or vaginal administration. Inpreferred embodiments, the final compounded formulations may beself-administered.

The kits of the invention may also contain a package which may becompartmentalized to receive in close confinement two or more containersof the invention. In some embodiments, the package may be box-like,being made of a moderately rigid material such as cardboard orreinforced paper. In other embodiments, the package may be a bag. Instill other embodiments, as described herein, there is no externalpackaging and all containers may be incorporated into one of thecontainers housing either an active or an inactive agent. This latterembodiment can be accomplished by securing containers such as pouches,sleeves or sacs, containing either active or inactive agents, as well asany mixing elements required for the compounding, to the interior of thelid of the main container. An individual skilled in the art can readilymodify the package to suit the individual needs of each kit and eachuse. The kits of the invention further contain instructions for theproper use of the components found therein.

The kits of the invention are intended for use in the treatment orprevention of a number of disorders in a variety of subjects includinghumans, dogs, cats, horses, fish, pigs, cows, sheep, deer, zoo animalsand laboratory animals (e.g., mice, rats, rabbits, monkeys, etc.). Theinvention intends to embrace unit of use kits containing the abovepreparations.

The following examples are offered by way of illustration, not by way oflimitation. While specific examples have been provided, the abovedescription is illustrative and not restrictive. Any one or more of thefeatures of the previously described embodiments can be combined in anymanner with one or more features of any other embodiments in the presentinvention. Furthermore, many variations of the invention will becomeapparent to those skilled in the art upon review of the specification.The scope of the invention should, therefore, be determined not withreference to the above description, but instead should be determinedwith reference to the appended claims along with their full scope ofequivalents.

All publications and patent documents cited in this application areincorporated by reference in pertinent part for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted. By their citation of various references in thisdocument, Applicants do not admit any particular reference is “priorart” to their invention.

EXAMPLES Example 1 Killing of Skin Cancer Cells

Sulindac enhances the killing of the human SCC-25 squamous cell skincancer cell line by tert-butyl hydroperoxide (TBHP). SCC-25 is a humansquamous cell carcinoma cell line developed by J G Rheinwald [Rheinwald,1981]. The cells are obtained from the American Type Culture Collection(ATCC # CRL-1628). The cells were maintained in minimum essential medium(Eagle) supplemented with 2 mM L-glutamine, Earle's BSS containing 1.5g/L sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodiumpyruvate and 10% fetal bovine serum.

The experimental approach was to pretreat SCC cells with sulindac for 24hr prior to exposure to TBHP for 2 hr. Sulindac (Sigma; S-8139) wasfreshly prepared as a IM stock solution in 1M Tris.Cl, pH 8.0. SCC cellswere suspended in culture medium containing 10% fetal bovine serum at acell density of 6×105 cells/ml. Cell suspensions containing 500 μMsulindac or no sulindac (Control) were plated in 96 well microtiterplates with a total of 6×104 cells per well. The plates were incubatedfor 24 hr at 37° C. in a 5% CO₂ incubator. The culture medium was thenremoved and the cells washed once with fresh culture medium withoutserum. After removal of the wash solution, fresh culture medium withoutserum that contained the indicated final concentration of TBHP was addedto the cells for 2 hrs. Serum was omitted from the medium during thetwo-hour exposure to TBHP since TBHP may be rapidly depleted in thepresence of serum.

Cell viability was determined by the CellTiter 96® Aqueous One CellProliferation Assay (Promega) according to the manufacture'sinstructions. The assay utilizes a novel tetrazolium compound(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium;MTS) and an electron-coupling reagent (phenazine ethosulfate; PES).Metabolically active cells convert the MTS to a water-soluble formazanby the action of cellular dehydrogenases. The amount of formazanproduced is measured by absorbance at 490 nm using a colorimetricmicrotiter plate reader (BioRad; Model 2550). Absorbance is directlyproportional to the number of viable cells.

Experiments to determine the toxicity of sulindac showed no detectableeffect on cell viability using the MTS assay at concentrations below 1mM. Therefore, a concentration of 500 μM sulindac was used for themajority of our experiments in cancer cell lines.

Experiments were performed with the SCC skin cancer cells that werepretreated with 500 μM sulindac using TBHP. The combination ofpretreatment with sulindac and oxidative stress by addition of TBHPenhanced the killing of the SCC carcinoma cells (FIG. 1). The decreasein viability of SCC cells pretreated with 500 μM sulindac was evident atconcentrations of TBHP from 100 μM to 700 μM.

FIG. 1 shows the effect of TBHP on viability of SCC skin cancer cellsfollowing pretreatment with 500 μM sulindac. SCC skin cancer cells wereincubated for 24 hr in the presence or absence of 500 μM sulindac. Themedium was removed and the cells washed once with fresh medium followedby addition of medium containing the indicated concentration of TBHP.After a 2 hr incubation at 37° C., the cells were assayed for viabilityas described.

Sulindac enhances the killing of the SCC skin cancer cell line byhydrogen peroxide: Experiments were performed with the SCC skin cancercells that were pretreated with 500 μM sulindac and then hydrogenperoxide (H₂O₂) for 24 hours instead of TBHP. The data clearly show thatthis alternative form of oxidative stress leads to a similar enhancedcell killing when cells are pretreated with sulindac (FIG. 2).

FIG. 2 shows the effect of hydrogen peroxide on viability of SCC skincancer cells following pretreatment with 500 μM Sulindac. SCC skincancer cells were incubated for 24 hr in the presence (♦) or absence (▪)of 500 μM sulindac. The medium was removed and the cells washed oncewith fresh medium followed by addition of medium containing theindicated concentration of hydrogen peroxide (H₂O₂). After a 24 hrincubation at 37° C., the cells were assayed for viability as described.

Sulindac enhances the killing of the SCC skin cancer cell line byarsenic trioxide: Experiments were performed with the SCC skin cancercells that were pretreated with 500 μM sulindac but using arsenictrioxide for 24 hours instead of peroxide. Arsenic trioxide was usedsince it is a compound capable of giving rise to oxidative damage by amechanism that leads to the generation of ROS inside the mitochondria.The data clearly show that this alternative form of oxidative stressleads to a similar enhanced cell killing when cells are pretreated withsulindac (FIG. 3).

FIG. 3 shows the effect of arsenic trioxide on viability of SCC skincancer cells. Following Pretreatment with 500 μM Sulindac. SCC skincancer cells were incubated for 24 hr in the presence or absence of 500μM sulindac. The medium was removed and the cells washed once with freshmedium followed by addition of medium containing the indicatedconcentration of arsenic trioxide. After a 24 hr incubation at 37° C.,the cells were assayed for viability as described.

Example 2 Killing of Colon Cancer Cells

Sulindac enhances the killing of the RKO colon cancer cell line bytert-butyl hydroperoxide (TBHP). RKO is a human colon carcinoma cellline developed by Michael Brattain [Brattain, 1984]. The cells wereobtained from the American Type Culture Collection (ATCC #CRL-2577). Thecells were maintained in minimum essential medium (Eagle) supplementedwith 2 mM L-glutamine, Earle's BSS containing 1.5 g/L sodiumbicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvateand 10% fetal bovine serum.

Experiments were performed with the RKO colon cancer cells that werepretreated with 500 μM sulindac using TBHP. The combination ofpretreatment with sulindac and oxidative stress by addition of TBHPenhanced the killing of the RKO carcinoma cells (FIG. 4). The decreasein viability of RKO cells pretreated with 500 μM sulindac wasstatistically “very significant” (P<0.005) at all concentrations of TBHPfrom 120 μM to 300 μM, the highest concentration tested.

FIG. 4 shows the effect of TBHP on viability of RKO colon cancer cells.Following Pretreatment with 500 μM Sulindac. RKO colon cancer cells wereincubated for 24 hr in the presence or absence of 500 μM sulindac. Themedium was removed and the cells washed once with fresh medium followedby addition of medium containing the indicated concentration of TBHP.After a 2 hr incubation at 37° C., the cells were assayed for viabilityas described. Error bars represent the SEM of four replicate samples.

Sulindac enhances the killing of the RKO colon cancer cell line byhydrogen peroxide: Experiments were performed with the RKO colon cancercells that were pretreated with 500 μM sulindac but using hydrogenperoxide (H₂O₂) instead of TBHP. The data clearly show that thisalternative form of oxidative stress leads to a similar enhanced cellkilling when cells are pretreated with sulindac (FIG. 5).

FIG. 5 shows the effect of hydrogen peroxide on viability of RKO coloncancer cells following pretreatment with 500 μM Sulindac. RKO coloncancer cells were incubated for 24 hr in the presence or absence of 500μM sulindac. The medium was removed and the cells washed once with freshmedium followed by addition of medium containing the indicatedconcentration of hydrogen peroxide (H₂O₂). After a 2 hr incubation at37° C., the cells were assayed for viability as described.

Example 3 Killing of Lung Cancer Cells

Sulindac enhances the killing of a lung carcinoma cell line by TBHP.Experiments using similar methods were performed with a lung carcinomacell line designated A549. The cell line is obtained from the AmericanType Culture Collection (ATCC #CCL 185). The line was established by D.J. Giard using an explant of lung carcinomatous tissue from a58-year-old Caucasian male [Giard, 1973]. Cells were grown in Ham's F12Kmedium supplemented with final concentrations of 2 mM L-glutamine, 1.6g/L sodium bicarbonate and 10% fetal bovine serum. Cultures weremaintained between 6×10³-6×10⁴ cells/cm² in standard tissue cultureflasks. The protocol for pretreatment of cells with 500 μM sulindacfollowed by a two hour exposure to various concentrations of TBHP wasthe same as that described for the RKO colon cancer cells.

Following a 24 hr pretreatment with 500 μM sulindac, the cells werewashed once to remove the excess sulindac before exposure to TBHP for 2hr. The lung cancer cell line also shows a marked decrease in cellviability when pretreated with 500 μM sulindac followed by exposure toTBHP (FIG. 6). Overall, the lung carcinoma cells appear to be moreresistant to TBHP, so higher concentrations were used compared to theexperiments with the RKO cells. At concentrations of 1 mM-2.5 mM TBHP,the difference in cell viability between control and sulindac treatedcells was statistically “extremely significant” (P<0.0001). In thepresence of 1.5-2.0 mM TBHP, approximately 50% of the untreated cellswere still viable whereas viability of cells pretreated with sulindacwas at background levels.

Our studies with lung cells reveal the same types of results as with theskin cells in terms of intracellular ROS levels. Lung cancer cellstreated with the combination of sulindac and TBHP had significantlyhigher levels of ROS than with either treatment alone. The results weresynergistic. Normal lung cells had lower levels of ROS with thecombination.

FIG. 6 shows the effect of TBHP on viability of lung cancer cellsfollowing pretreatment with 500 μM Sulindac. Lung cancer cells wereincubated for 24 hr in the presence or absence of 500 μM sulindac. Themedium was removed and the cells washed once with fresh medium followedby addition of medium containing the indicated concentration of TBHP.After a 2 hr incubation at 37° C., the cells were assayed for viabilityas described. Error bars represent the SEM of four replicate samples.

Sulindac enhances the killing of a lung carcinoma cell line by hydrogenperoxide: Experiments were performed with the lung carcinoma cells usinghydrogen peroxide instead of TBHP. Lung cancer cells pretreated with 500μM sulindac are differentially sensitive to killing by H₂O₂ compared tountreated cells (FIG. 7). In the range of 2.5-5.0 mM H₂O₂, thedifference in cell viability between sulindac-treated and untreatedcells is statistically “highly significant” (P<0.001).

FIG. 7 shows the effect of hydrogen peroxide on viability of lung cancercells following pretreatment with 500 μM sulindac. Lung cancer cellswere incubated for 24 hr in the presence or absence of 500 μM sulindac.The medium was removed and the cells washed once with fresh mediumfollowed by addition of medium containing the indicated concentration ofhydrogen peroxide. After a 2 hr incubation at 37° C., the cells wereassayed for viability as described. Error bars represent the SEM forreplicate samples.

Example 4 Killing of Melanoma Cells

Sulindac enhances the killing of the human WM-266-4 Melanoma skin cellline by tert-butyl hydroperoxide (TBHP). WM-266-4 is a human melanomacell line developed by the Wistar Institute. The cells are obtained fromthe American Type Culture Collection (ATCC # CRL-1676). The cells weremaintained in minimum essential medium (Eagle) supplemented with 2 mML-glutamine, Earle's BSS containing 1.5 g/L sodium bicarbonate, 0.1 mMnon-essential amino acids, 1.0 mM sodium pyruvate and 10% fetal bovineserum.

Experiments were performed with the Melanoma skin cancer cells that werepretreated with 500 μM sulindac using TBHP. The combination ofpretreatment with sulindac and oxidative stress by addition of TBHPenhanced the killing of the Melanoma cells (FIG. 8). The decrease inviability of Melanoma cells pretreated with 500 μM sulindac was evidentat concentrations of TBHP from 600 μM to 2000 μM.

FIG. 8 shows the effect of TBHP on viability of melanoma cells followingpretreatment with 500 μM Sulindac. Melanoma skin cancer cells wereincubated for 24 hr in the presence or absence of 500 μM sulindac. Themedium was removed and the cells washed once with fresh medium followedby addition of medium containing the indicated concentration of TBHP.After a 2 hr incubation at 37° C., the cells were assayed for viabilityas described.

Sulindac enhances the killing of the human WM-266-4 Melanoma skin cellline by arsenic trioxide: Experiments were performed with the melanomacells that were pretreated with 500 μM sulindac but using arsenictrioxide for 24 hours instead of TBHP. Arsenic trioxide was used sinceit is a compound capable of giving rise to oxidative damage by amechanism that leads to the generation of elevated ROS levels inside themitochondria. The data clearly show that this alternative form ofoxidative stress leads to a similar enhanced cell killing effect whencells are pretreated with sulindac (FIG. 9).

FIG. 9 shows the effect of arsenic trioxide on viability of melanomacells following pretreatment with 500 μM Sulindac. Melanoma cells wereincubated for 24 hr in the presence or absence of 500 μM sulindac. Themedium was removed and the cells washed once with fresh medium followedby addition of medium containing the indicated concentration of arsenictrioxide. After a 24 hr incubation at 37° C., the cells were assayed forviability as described. Sulindac does not enhance the killing of normalcells by TBHP or arsenic trioxide.

Example 5 No Effects on Normal Human Skin Cells

Experiments using similar methods were performed with primary humanneonatal foreskin epidermal keratinocytes. These cells were oflow-passage and purified using previously accepted protocols (providedby J. Li, University of Miami, Fl.). Cells were grown in 50%Keratinocyte-SFM media [Gibco-Invitrogen (Catalog #17005-042)supplemented with human recombinant Epidermal Growth Factor 1-53 (EGF1-53) and Bovine Pituitary Extract (BPE)] with 50% EpiLife medium[Cascade Biologics (Catalog #Epi-500-Ca)] supplemented with purifiedbovine serum albumin, purified bovine transferrin, hydrocortisone,recombinant human insulin-like growth factor type-1 (rhIGF-1),prostaglandin E2 (PGE2), and recombinant human epidermal growth factor(rhEGF).

Experimental conditions: Normal HEK skin cells plated onto 6-well platesat cell density of 60-70% confluence. Sulindac (0.5 mM)×24 hours (celldensity remained about the same) followed by TBHP×2 hours followed byROS indicator dye (5 μM)×1 hour (Table 1).

TABLE 1 % Cells with ROS Uptake by Fluorescence (fold increase comparedto untreated control) Sul + TBHP Sul + TBHP Sul + TBHP No Sul/No TBHPSul alone (200 μM) (400 μM) (800 μM) 3 2 3(0) 12(4) 68(23) 2(0)  5(2)60(20)

Sulindac had no significant effect when combined with TBHP on the levelof ROS uptake in normal HEK cells.

The combination of pretreatment with sulindac and oxidative stress byaddition of TBHP or arsenic trioxide had no significant effect on theviability of the normal human epidermal keratinocytes (FIG. 10). Asimilar effect was seen with hydrogen peroxide. These results are incontrast to what was evident with the skin cancer cells (FIGS. 1-3). Atthe lower concentrations of TBHP (50-400 μM), in combination withsulindac, there was an enhanced resistance to oxidative stressmanifested by a protective effect. This protective effect was lost atconcentrations of TBHP above 400 μM.

FIG. 10 shows the effect of TBHP on viability of normal human epidermalkeratinocytes following pretreatment with 500 μM Sulindac. Normal humanepidermal keratinocyte cells were incubated for 24 hr in the presence orabsence of 500 μM sulindac. The medium was removed and the cells washedonce with fresh medium followed by addition of medium containing theindicated concentration of (a) TBHP or (b) arsenic trioxide. After a 2hr incubation with TBHP or a 24-hour incubation with arsenic trioxide at37° C., the cells were assayed for viability as described.

Normal Human Colon & Lung Cells: Pretreatment of normal human colon andlung cells with sulindac provides protection from oxidative stress.CCD-18Co is a normal human colon cell line and MRC-5 is a normal humanlung cell line obtained from the American Type Culture Collection (ATCC#CRL-1459 and #CCL-171). The cells were maintained in minimum essentialmedium (Eagle) supplemented with 2 mM L-glutamine, Earle's BSScontaining 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids,1.0 mM sodium pyruvate and 10% fetal bovine serum.

Experiments were performed with the normal human colon and normal humanlung cells that were pretreated with 500 μM sulindac followed by the useof TBHP. The combination of pretreatment with sulindac and oxidativestress by addition of TBHP provided protection from oxidative stress.(FIGS. 11 & 12).

FIG. 11 shows the effect of TBHP on normal human colon cells pretreatedwith sulindac. Normal diploid colon cells were incubated for 24 hr withmedium containing 500 μM sulindac or no addition (no sulindac). Cellswere washed free of sulindac prior to incubation for 2 hr with theindicated concentration of TBHP. Cell viability was measured using theMTS assay. Results are expressed as % of Control (cells that were notpretreated or exposed to TBHP). Error bars are standard error of themean (SEM) expressed as a % of the mean value of four replicate samples.

FIG. 12 shows the effect of TBHP on normal human lung cells pretreatedwith sulindac. Normal diploid lung cells were incubated for 24 hr withmedium containing 500 μM sulindac or no addition (no sulindac). Cellswere washed free of sulindac prior to incubation for 2 hr with theindicated concentration of TBHP. Cell viability was measured using theMTS assay described. Results are expressed as % of Control (cells thatwere not pretreated or exposed to TBHP). Error bars are standard errorof the mean (SEM) expressed as a % of the mean value of four replicatesamples.

In sharp contrast to the results obtained with the transformed cells,each of the three normal human cell lines (skin, colon, and lung) showedenhanced resistance to oxidative stress when pretreated with sulindacfor 24 hr.

Normal Cardiac & Retinal Cells: Sulindac does not enhance the killing ofnormal cardiac and retinal cells by hydrogen peroxide and TBHP. Primaryneonatal rat cardiac myocytes and ARPE-19, a human retinal pigmentepithelial cell line that is not fully transformed, were used in similarexperiments with sulindac followed by peroxide. The results showed thatthere was no enhanced killing of these normal cells using thecombination of sulindac and peroxide.

FIG. 16 shows that at a constant non-toxic TBHP concentration incombination with escalating doses of sulindac, the effects on normalversus cancer cells can be better elucidated. There is a protectiveeffect on normal cells while an enhanced killing of cancer cells occurs.

In this experiment, the gap or difference in cell survival widensbetween the cell types over a 3-5× concentration range of sulindac (500μM-1500 μM).

Example 6 Sulindac Metabolites/Derivatives

A sulindac metabolite enhances the killing of cancer cells by TBHP. Thestructure of sulindac and its metabolites and a derivative, sulindacmethionine sulfoxide (SMO), are shown in FIG. 13. A metabolite ofsulindac, specifically sulindac sulfone, was effective in enhancing thekilling of skin cancer cells (SCC-25) when exposed to TBHP (FIG. 14).Sulindac was more potent than the sulfone metabolite. The SMO derivativeand the other metabolite of sulindac, specifically sulindac sulfide,were not effective in enhancing the killing of the skin cancer cellswhen exposed to TBHP.

However, when lung cancer cells or melanoma cells were exposed to TBHP,the sulindac sulfide metabolite had activity in reducing the cellviability. In summary, in all cancer cell studies, sulindac and sulindacsulfone had the most potent killing effect (FIG. 15).

FIG. 14 shows the results from skin cancer cells treated with sulindac,sulindac metabolites, or a sulindac derivative. Skin cancer cells wereincubated for 24 hr with medium containing 500 μM sulindac, 500 μMsulindac sulfone, 500 μM SMO, 50 μM sulindac sulfide or no addition (nopretreatment). Cells were washed free of compound prior to incubationfor 2 hr with the indicated concentration of TBHP. Cell viability wasmeasured using the MTS assay described.

FIG. 15 shows lung cancer cells treated with sulindac or itsmetabolites. Lung cancer cells were incubated for 24 hr with mediumcontaining 500 μM sulindac, 250 μM sulindac sulfone, 250 μM sulindacsulfide or no addition (no pretreatment). Cells were washed free ofsulindac prior to incubation for 2 hr with the indicated concentrationof TBHP. Cell viability was measured using the MTS assay. Results areexpressed as % of Control (cells that were not pretreated or exposed toTBHP). Error bars are standard error of the mean (SEM) expressed as a %of the mean value of four replicate samples.

Other experiments: SCC skin cancer cells plated onto 6-well plates atcell density of 50-60% confluence. Sulindac (0.5 mM)×24 hours (celldensity increased to 80-90% confluence) followed by TBHP (100 μM)×2hours followed by ROS indicator dye (5 μM)×1 hour (Table 2).

TABLE 2 Experimental % Cells with ROS fold increase compared ConditionUptake by Fluorescence to untreated control NO Sul/NO TBHP 1.6%  — Sulalone  3% 2 TBHP alone 11% 7 Sul + TBHP 30% 19

Conclusion: Sulindac enhances the levels of ROS uptake in SCC skincancer cells. Sulindac in combination with TBHP geometrically enhancesthe levels of ROS uptake. Sulindac tends to increase the number ofspindle cells in the culture compared to untreated controls

Other NSAIDS do not enhance the killing of the SCC-25 skin cancer cellsor RKO colon carcinoma cell line by TBHP. Acetylsalicylic acid(Aspirin), ibuprofen (Motrin), diclofenac sodium (Voltaren/Solaraze),and celecoxib (Celebrex) at comparable active concentrations, were foundto be ineffective compared to sulindac in sensitizing the skin cancercells and or colon cancer cells to killing by TBHP.

Sulindac in combination with an oxidizing agent increases theintracellular levels of ROS in cancer cells. SCC skin cancer cells wereplated onto 6-well plates at a cell density of 50-60% confluence. Thecultures were pretreated with sulindac (500 uM) for 24 hours followed bythe addition of TBHP (100 uM) for two hours. During this period of time,the cell density in the 6-well plates increased to 80-90% confluent.This was immediately followed by the addition of the ROS indicator dye[(5 uM) Carboxy-H2DCFDA (5-(and6-)carboxy-2′,7′-dichlorodihydrofluorescein diacetate, Molecular Probes,Carlsbad, Calif.] for one hour. The cancer cells were visually inspectedusing a fluorescence microscope and counted. Cells with greenfluorescence were considered to have an increased level of ROS. Sulindacand TBHP both enhanced the ROS levels in SCC cells (Table 3). Sulindacin combination with TBHP synergistically enhanced the ROS levels in skincancer cells (2× the additive value of sulindac or TBHP alone).

TABLE 3 % Cells containing fold increase (compared Treatment ROS* tountreated control) NO Sul/NO TBHP 1.6 — Sul (500 μM) alone 3 2 TBHP (100μM) alone 11 7 Sul (500 μM) + TBHP (100 μM) 30 19 *ROS levels weremeasured by green fluorescence observed using a fluorescence microscope.

Sulindac in combination with an oxidizing agent has no effect on theintracellular levels of ROS in normal skin cells. Human neonatalepidermal keratinocytes were plated into 6-well plates at a cell densityof 60-70% confluence. The cultures were pretreated with sulindac (500uM) for 34 hours followed by the addition of different concentrations ofTBHP for 2 hours. During this period of time, the cell density in the6-well plates remained the same (there was no increase in cellproliferation). This was followed by the addition of the ROS indicatordye. Sulindac when combined with TBHP had no significant effect overwhat was seen with sulindac or TBHP alone on the ROS levels in normalhuman skin cells (Table 4).

TABLE 4 % Cells containing ROS* (fold increase compared to Treatmentuntreated control) No Sul/No TBHP 3 (0) Sul (500 uM) alone 2 (0) TBHP(200 uM) alone 2 (0) TBHP (400 uM) alone 5 (2) TBHP (800 uM) alone 60(20) Sul (500 uM) + TBHP (200 uM) 3 (0) Sul (500 uM) + TBHP (400 uM) 12(4)  Sul (500 uM) + TBHP (800 uM) 68 (23) ROS levels were measured bygreen fluorescence observed using a fluorescence microscope

Additional information on the experiments with sulindac and the ROSgenerators (hydrogen peroxide, TBHP and arsenic): Sulindac waspre-incubated for various times prior to the addition of the oxidant.Time course studies revealed that it was necessary for the sulindac tobe pre-incubated for a minimum of 6 hours to see an effect. Optimumeffects were seen with an 18-24 hour incubation. There was no differencein the results with longer time courses such as 48-72 hours. After thepre-incubation with sulindac, the cells were washed and the oxidant wasadded with or without sulindac.

TBHP experiments were performed using 2 hour incubations with noadditional sulindac.

Hydrogen peroxide and arsenic trioxide experiments were performed with24 hour incubations with the re-addition of sulindac.

Patients with precancerous growths and BCC's exhibited a beneficialresponse to a topical formulation of a composition of sulindac andhydrogen peroxide. A few drops of a topical formulation of 5% or 10%sulindac gel was administered once or twice a day for up to 4 weeks toseveral patients over skin areas that had precancerous growths and/orBCC's. After 10-15 minutes, this application was followed by theapplication of several drops of hydrogen peroxide solution (6%-25%) for5-10 minutes once or twice a day. In each instance, after 2-5 days, thepatients exhibited some minimal swelling and redness with mildirritation to the involved areas not seen on normal adjacent skin. Abright yellowish hue was localized on the growths that seemed to be mostintense on the overlying scale. After 4 weeks of treatment, a number ofprecancerous growths were visibly diminished in size or gone. The BCC'slooked much smaller in size and, in certain locations, were no longervisible. There has been no recurrence of the growths that have visiblydisappeared in the areas of treatment after 6 months. The patients arecontinued to be followed for an evaluation of their long-term responses.

Example 7 Treatment of Patient with Actinic Keratoses

A male patient had a number of scaly growths (actinic keratoses), on theface. Four areas were selected for treatment, including one on histemple with a large scale crust. After continuous treatment with the 5%sulindac gel formulation, in combination with 6% hydrogen peroxidesolution at least once a day for 4 weeks, the patient had a completeresponse. All four actinic keratoses resolved and disappeared, includingthe one with the large scale. There has been no recurrence in the areasof the treated growths after 6 months of observation.

Example 8 Treatment of Patient with Basal Cell Carcinoma

A male in his mid to late 50's had recurrent basal cell carcinoma on theright temple and right forehead. Doctors recommended their removal byMoh's surgery. However, the surgery could have led to unacceptablecosmetic results with scarring. After continuous treatment with 10%sulindac gel formulation in combination with 12% hydrogen peroxidesolution, for at least once a day for 10 weeks, the patient had acomplete response. Both skin cancers are not visible after 3 months offollow-up. (See, FIGS. 17 and 18).

FIG. 17 is a scan of a photograph showing basal cell carcinomas beforetreatment with sulindac peroxide. The black arrows point to tworecurrent basal cell carcinomas on the face of a patient. A 0.5centimeter ulcerated area with pearly irregular borders on the rightforehead near the right upper eyelid and a 1.5 centimeter poorlycircumscribed lesion with pearly ill-defined nodular borders on theright temple area near the hairline.

FIG. 18 is a scan of a photograph showing the basal cell carcinomas ofFIG. 17, after treatment with sulindac peroxide. The black arrows pointto the two areas on the skin of the face that previously containedbiopsy proven recurrent basal cell skin cancers (as described in FIG.17) showing the disappearance of the cancers after treatment withsulindac-peroxide formulations. The areas have healed with small amountsof scar tissue present.

Example 9 Treatment of Patient with Squamous Cell Carcinoma

A male in his 60's had a non-healing large scaly growth on his righthand. It was a biopsy proven hypertrophic actinic keratosis. The growthhad been treated with liquid nitrogen three times over the previous yearwithout success. On visual inspection, the growth could have representeda superficial squamous cell carcinoma. After the application of thetopical 10% sulindac gel in combination with 25% hydrogen peroxidesolution (Table 5), for at least once a day for 6 weeks, the patient hada complete response. (See, FIGS. 19 and 20). The growth has not recurredafter 3 months of follow-up).

FIG. 19 is a scan of a photograph showing a hyperkeratotic lesion beforetreatment with the sulindac-peroxide formulation. The white arrow pointsto a 1 centimeter hyperkeratotic lesion on the right hand representing ahypertrophic actinic keratosis or squamous cell carcinoma beforetreatment with sulindac-peroxide formulations.

FIG. 20 is a scan of a photograph showing the hyperkeratotic lesionafter treatment with sulindac-peroxide of the same patient in FIG. 19.The white arrow points to the area of previous skin lesion (as describedin FIG. 19) showing the eradication of the condition after treatmentwith the sulindac-peroxide formulations. Only a small amount of scartissue with minimal scaling remains.

TABLE 5 Formulation of topical 5% sulindac gel. Ingredient % Deionizedwater 71.08 SD Alcohol 40 12.00 KOH 10.00 Sulindac 5.00 Hydroxy methylcellulose 1.00 Xantham gum 0.50 Glydant Plus 0.20 Citric Acid 0.20Disodium EDTA 0.02

This base formula is used for 10% sulindac gels, 20% sulindac gels, 50%sulindac gels. Variations of the formula are easily adapted by one ofordinary skill in the art.

Pre-clinical experience in human patients: Sulindac gel and hydrogenperoxide gel or liquid were applied using calibrated dropper bottlesdirectly on the skin of volunteers with actinic keratoses. Sulindac wasgenerally applied first although in a few instances, the peroxide wasapplied first. The sulindac was applied and allowed to dry for 5-10minutes followed by the application of the peroxide. The products werenot washed off the skin until after 8-12 hours of applications. Productswere applied twice a day to each individual area.

Example 10 R- and S-Epimers

The R- and S-epimers of sulindac can be separated using chiral columnsand HPLC. The sulindac epimers were separated and isolated from theracemic mixture with scale-up of sufficient quantities for our studiesusing a preparative chiral column. The chiral column (R, R)-Welk-01chiral column (25 cm×4.6 mm) was purchased from Regis Technologies(Milton Grove, Ill.). As shown in FIG. 22, the 2 epimers of sulindacwere separated quite well, under the conditions described in the legendof FIG. 22. Two peaks were observed eluting after 22.5 and 28 min. Thepeak tubes were combined and the solvent evaporated. The separatedepimers were then dissolved in 1M Tris Cl pH 7.4. In order to determinewhich epimer was present in each peak, the material in each peak wasincubated with Escherichia coli MsrA and 15 mM DTT, which would convertthe S-epimer to sulindac sulfide. At the end of the incubation, thereduced product was extracted into benzene and the optical density readat 350 nm as previously described (Etienne F, Resnick L, Sagher D, BrotN, Weissbach H. Biochem Biophys Res Comm. 2003; 312:1005-10). It wasdetermined that the peak eluting at 28 minutes was the S-epimer ofsulindac.

The R-epimer of sulindac is poorly converted to the sulfide (COXinhibitor) in whole normal cells as compared to the S-epimer: Theindividual sulindac epimers were evaluated for their conversion to thesulfide in cultured whole normal cells, employing an assay developed inthis laboratory (Etienne F, Resnick L, Sagher D, Brot N, Weissbach H.Biochem Biophys Res Comm. 2003; 312:1005-10). Enriched cultures ofneonatal rat cardiac myocytes were obtained from 1-2 day-old neonatalrats by stepwise trypsin dissociation and plated at a density of 104cells/60 mm dish in DMEM supplemented with 5% fetal calf serum,penicillin, and streptomycin. After 3-5 days, the cells were transferredto a defined serum-free DMEM/M-199 (4:1) medium supplemented withtransferring, vitamin B12 and insulin. The final cultures containedgreater than 97% cardiac myocytes contracting at greater than 200 beatsper minute. To inhibit fibroblast growth, 0.1 mM bromodeoxyuridine wasincluded in the medium for the first 3-5 days after plating. Cellcultures are incubated with sulindac or its individual epimers forspecified times.

After the incubations, the cells were pelleted by low speedcentrifugation and lysed by the addition of 200 μl of 100% acetonitrile,followed by vigorous vortexing. The lysate was centrifuged for 15minutes at 14,000×g to pellet cell debris. 50 μl of the supernatant wastransferred to an HPLC autosampler microvial. The separation ofmetabolites is carried out on a 4.6×75 mm C18 reverse-phase column. Anisocratic flow of 1 ml/min consisting of 50% 50 mM sodium acetate, pH4.73 and 50% acetonitrile was utilized. The metabolites are detected ata wavelength of 330 nm. With the Waters model 2487 detector, pmolequantities of the sulfide can be detected.

As shown in FIG. 23, in normal cardiac myocytes, the R-epimer ofsulindac was very poorly converted to the sulfide (greater than 10-foldless conversion) compared to the S-epimer, under the conditionsdescribed in the legend.

Table 6 below, shows that two normal cell lines (lung andcardiomyocytes) do not reduce the R epimer as efficiently as the Sepimer (<10% R/S). However, in 3 cancer cell lines (Lung, HeLa, and skinSCC), there is an enhanced conversion of the R-epimer (greater than40%), unlike that found in normal cells (less than 10%). A significantreduction of the R-epimer of sulindac, although not seen in normalcells, has been observed in some malignant cells.

The cells are incubated with 200 μM R- or S-Sulindac epimer for 4 hoursin a defined, serum-free medium (EX-CELL®). After incubation, the cellsare collected, rinsed and lysed with acetonitrile. The cytosoliccontents are separated on a C-18 column.

TABLE 6 Amount Formed (picomoles) Cell R-epimer S-epimer R/S (%) CancerLung 7.5 17.7 42 HeLa 9.3 7.9 118 Skin 35.3 57 62 Normal Lung 2.2 35 6Cardiomyocyte 11.6 120.9 9

The R- and S-epimers of sulindac enhance the killing of SCC skin andlung cancer cells by TBHP or hydrogen peroxide: Milligram quantities ofeach epimer were isolated (as shown in FIG. 22) and tested for theirability to selectively kill cancer cells. The SCC skin cancer cells orlung cancer cells were pretreated with 500 μM sulindac or 500 μMR-sulindac or 500 μM S-sulindac for 24 hours followed by the addition ofTBHP for 2 hours (or hydrogen peroxide for 24-hours). The combination ofpretreatment with sulindac or its individual epimers and oxidativestress enhanced the killing of the SCC and lung carcinoma cells. Asshown in FIGS. 24 and 25, both epimers of sulindac had cancer killingeffects that were comparable in potency to the sulindac racemic mixture.These results indicate that the R-epimer of sulindac has the potentialof selectively killing cancer cells. Since in normal cells, but notcancer cells, the R-epimer of sulindac is not efficiently converted tothe sulfide, the active COX inhibitor, it would be expected that normalcells would have a lower toxicity profile.

We have used a chiral column to separate the two epimers of sulindac andhave evidence for the identification of the resolved HPLC peaks. TheR-epimer of sulindac retains its ability to selectively kill cancercells, while showing little or no conversion in whole normal cells tothe sulfide (COX inhibitor). These results indicate that the R-epimer ofsulindac has biochemical and pharmacological properties that would makeit a better therapeutic agent than the unresolved sulindac-R, S mixture.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims.

1. A pharmaceutical composition comprising: sulindac, sulindacmetabolites, sulindac derivatives, R- and S-epimers thereof and anoxidating agent, wherein the oxidizing agent concentration is in a rangeof about 1% to 50% by weight and the sulindac concentration, and/orR-epimers thereof, is in range from about 1% to 50% by weight.
 2. Thepharmaceutical composition of claim 1, wherein the sulindac metabolitecomprises sulindac sulfide or sulindac sulfone and R- and S-epimersthereof.
 3. The pharmaceutical composition of claim 1, wherein thesulindac derivative comprises sulindac methionine sulfoxide and R- andS-epimers thereof.
 4. The pharmaceutical composition of claim 1, whereinthe oxidating agent comprises at least one of: peroxides, nitrates,nitrites, perchlorates, chlorates, chlorites, hypochlorite, dichromates,permanganates, and persulfates, bromine, arsenic trioxide, retinoic acid(and its derivatives), K antimonyl tartrate, doxorubicin, imexon, andbortezomib, hydrogen peroxide; inorganic peroxides, sodium peroxyboratetetrahydrate (sodium perborate tetrahydrate) calcium peroxide; peroxidecomplexes, urea hydrogen peroxide; superoxide salts, sodium superoxide;superoxide free radical (O₂*) dismutates; organic peroxides,hydroperoxides (ROOH), lipid hydroperoxides, artemisinin and derivativesthereof; 1-Hydroperoxycyclohexyl-1-hydroxy cyclohexyl peroxide,tert-Butyl hydroperoxide; fatty acid hydroperoxides, linolenic acidhydroperoxide, arachidonic acid hydroperoxide, docosahexaenoic acidhydroperoxide, cholesterol hydroperoxide, cholesteryl linoleatehydroperoxide, trilinolein hydroperoxide, phosphatidylcholinehydroperoxide, phosphatidylethanolamine hydroperoxide;3-chloroperoxybenzoic acid, 1,1-bis(tert-butylperoxy)cyclohexane,peracetic acid, monoperoxyphthalic acid, tert-butyl peroxide,2,5,bis(tert-butylperoxy)-2,5-dimethyl-3-hexyne, cumene hydroperoxide,2,5-bis(tert-butylperoxy)-2,5-dimethylhexane, lauroyl peroxide, benzoylperoxide, dicumyl peroxide, 2,5-dihydroperoxy-2,5-dimethylhexane,tert-butyl peracetate, tert-amyl hydroperoxide, diisononanoyl peroxide,decanoyl peroxide, succinic acid peroxide, 2,4-pentanedione peroxide,di-tert-butyl peroxide, tert-butyl cumyl peroxide,bis(t-butylperoxy)-diisopropylbenzene, and1-((hydroperoxycyclohexyl)dioxy)-cyclohexanol; hydrogen peroxide,compounds containing a peroxy (peroxo) —O—O— moiety, superoxides,peroxide precursor compounds; endoperoxides, diacyl peroxides, ketoneperoxides, peroxydicarbonates, peroxyesters, dialkyl peroxides,peroxyketals, and peroxyacids.
 5. The pharmaceutical composition ofclaim 1, wherein the oxidating agent comprises hydrogen peroxide,tert-butyl hydroperoxide (TBHP), and arsenic trioxide.
 6. A topicalpharmaceutical composition comprising a sulindac gel and R- andS-epimers thereof, and an oxidizing agent.
 7. The topical pharmaceuticalcomposition of claim 6, wherein a 5% sulindac gel comprises: 71.08%deionized water; 12.00% SD Alcohol 40; 10% KOH; 5% Sulindac and/or R-and S-epimers thereof; 1% Hydroxy methyl cellulose; 0.50% Xantham gum;0.20% Glydant Plus; 0.20% Citric Acid; and 0.02% disodium EDTA.
 8. Thepharmaceutical topical composition of claim 6, wherein the sulindac geland R- and S-epimers thereof, are in the range of 1% to 50% sulindac byweight.
 9. The pharmaceutical topical composition of claim 6, whereinthe oxidating agent comprises hydrogen peroxide, tert-butylhydroperoxide (TBHP), and arsenic trioxide and the oxidizing agentconcentration is in a range of about 1% to 50% by weight and thesulindac concentration, and R- and S-epimers thereof, is in range fromabout 1% to 50% by weight.
 10. The pharmaceutical topical composition ofclaim 6, wherein the sulindac gel comprises 10% sulindac and 25%hydrogen peroxide.
 11. A method of treating an abnormal cell, saidmethod comprising administering to an abnormal cell a pharmaceuticalcomposition comprising sulindac, or sulindac metabolites, or sulindacderivatives or combinations thereof, and R- and S-epimers thereof, andan oxidating agent, thereby treating an abnormal cell.
 12. The method ofclaim 11, wherein the sulindac metabolite comprises sulindac sulfide orsulindac sulfone and R- and S-epimers thereof.
 13. The method of claim11, wherein the sulindac derivative is sulindac methionine sulfoxide andR- and S-epimers thereof.
 14. The method of claim 11, wherein theoxidating agent comprises at least one of: peroxides, nitrates,nitrites, perchlorates, chlorates, chlorites, hypochlorite, dichromates,permanganates, and persulfates, bromine, arsenic trioxide, retinoic acid(and its derivatives), K antimonyl tartrate, doxorubicin, imexon, andbortezomib, hydrogen peroxide; inorganic peroxides, sodium peroxyboratetetrahydrate (sodium perborate tetrahydrate) calcium peroxide; peroxidecomplexes, urea hydrogen peroxide; superoxide salts, sodium superoxide;superoxide free radical (O₂*) dismutates; organic peroxides,hydroperoxides (ROOH), lipid hydroperoxides, artemisinin and derivativesthereof; 1-Hydroperoxycyclohexyl-1-hydroxy cyclohexyl peroxide,tert-Butyl hydroperoxide; fatty acid hydroperoxides, linolenic acidhydroperoxide, arachidonic acid hydroperoxide, docosahexaenoic acidhydroperoxide, cholesterol hydroperoxide, cholesteryl linoleatehydroperoxide, trilinolein hydroperoxide, phosphatidylcholinehydroperoxide, phosphatidylethanolamine hydroperoxide;3-chloroperoxybenzoic acid, 1,1-bis(tert-butylperoxy)cyclohexane,peracetic acid, monoperoxyphthalic acid, tert-butyl peroxide,2,5,bis(tert-butylperoxy)-2,5-dimethyl-3-hexyne, cumene hydroperoxide,2,5-bis(tert-butylperoxy)-2,5-dimethylhexane, lauroyl peroxide, benzoylperoxide, dicumyl peroxide, 2,5-dihydroperoxy-2,5-dimethylhexane,tert-butyl peracetate, tert-amyl hydroperoxide, diisononanoyl peroxide,decanoyl peroxide, succinic acid peroxide, 2,4-pentanedione peroxide,di-tert-butyl peroxide, tert-butyl cumyl peroxide,bis(t-butylperoxy)-diisopropylbenzene, and1-((hydroperoxycyclohexyl)dioxy)-cyclohexanol; hydrogen peroxide,compounds containing a peroxy (peroxo) —O—O— moiety, superoxides,peroxide precursor compounds; endoperoxides, diacyl peroxides, ketoneperoxides, peroxydicarbonates, peroxyesters, dialkyl peroxides,peroxyketals, and peroxyacids.
 15. The method of claim 11, wherein theoxidating agent wherein the oxidating agent comprises hydrogen peroxide,tert-butyl hydroperoxide (TBHP), and arsenic trioxide.
 16. The method ofclaim 11, wherein the composition comprises a sulindac gel and anoxidizing agent.
 17. The method of claim 16, wherein a 5% sulindac gelcomprises: 71.08% deionized water; 12.00% SD Alcohol 40; 10% KOH; 5%Sulindac and/or R- and S-epimers thereof; 1% Hydroxy methyl cellulose;0.50% Xantham gum; 0.20% Glydant Plus; 0.20% Citric Acid; and 0.02%disodium EDTA.
 18. The method of claim 16, wherein the sulindac gel isin the range of 1% to 50% sulindac by weight.
 19. A method of treatingcancer, said method comprising administering to a cancer cell apharmaceutical composition comprising sulindac, or sulindac metabolites,or sulindac derivatives or combinations thereof, and/or R- and S-epimersthereof, and an oxidating agent, thereby treating cancer.
 20. The methodof claim 19, wherein the sulindac metabolite comprises sulindac sulfideor sulindac sulfone.
 21. The method of claim 19, wherein the oxidatingagent comprises at least one of: peroxides, nitrates, nitrites,perchlorates, chlorates, chlorites, hypochlorite, dichromates,permanganates, and persulfates, bromine, arsenic trioxide, retinoic acid(and its derivatives), K antimonyl tartrate, doxorubicin, imexon, andbortezomib, hydrogen peroxide; inorganic peroxides, sodium peroxyboratetetrahydrate (sodium perborate tetrahydrate) calcium peroxide; peroxidecomplexes, urea hydrogen peroxide; superoxide salts, sodium superoxide;superoxide free radical (O₂*) dismutates; organic peroxides,hydroperoxides (ROOH), lipid hydroperoxides, artemisinin and derivativesthereof; 1-Hydroperoxycyclohexyl-1-hydroxy cyclohexyl peroxide,tert-Butyl hydroperoxide; fatty acid hydroperoxides, linolenic acidhydroperoxide, arachidonic acid hydroperoxide, docosahexaenoic acidhydroperoxide, cholesterol hydroperoxide, cholesteryl linoleatehydroperoxide, trilinolein hydroperoxide, phosphatidylcholinehydroperoxide, phosphatidylethanolamine hydroperoxide;3-chloroperoxybenzoic acid, 1,1-bis(tert-butylperoxy)cyclohexane,peracetic acid, monoperoxyphthalic acid, tert-butyl peroxide,2,5,bis(tert-butylperoxy)-2,5-dimethyl-3-hexyne, cumene hydroperoxide,2,5-bis(tert-butylperoxy)-2,5-dimethylhexane, lauroyl peroxide, benzoylperoxide, dicumyl peroxide, 2,5-dihydroperoxy-2,5-dimethylhexane,tert-butyl peracetate, tert-amyl hydroperoxide, diisononanoyl peroxide,decanoyl peroxide, succinic acid peroxide, 2,4-pentanedione peroxide,di-tert-butyl peroxide, tert-butyl cumyl peroxide,bis(t-butylperoxy)-diisopropylbenzene, and1-((hydroperoxycyclohexyl)dioxy)-cyclohexanol; hydrogen peroxide,compounds containing a peroxy (peroxo) —O—O— moiety, superoxides,peroxide precursor compounds; endoperoxides, diacyl peroxides, ketoneperoxides, peroxydicarbonates, peroxyesters, dialkyl peroxides,peroxyketals, and peroxyacids.
 22. The method of claim 19, wherein theoxidating agent wherein the oxidating agent comprises hydrogen peroxide,tert-butyl hydroperoxide (TBHP), and arsenic trioxide and the oxidizingagent concentration is in a range of about 1% to 50% by weight.
 23. Apharmaceutical composition comprising: R-epimers of sulindac, sulindacderivatives, and an oxidating agent; wherein the oxidizing agentconcentration is in a range of about 1% to 50% by weight and thesulindac concentration, and R-epimers thereof, is in range from about 1%to 50% by weight.
 24. The pharmaceutical composition of claim 23,wherein the oxidating agent comprises at least one of: peroxides,nitrates, nitrites, perchlorates, chlorates, chlorites, hypochlorite,dichromates, permanganates, and persulfates, bromine, arsenic trioxide,retinoic acid (and its derivatives), K antimonyl tartrate, doxorubicin,imexon, and bortezomib, hydrogen peroxide; inorganic peroxides, sodiumperoxyborate tetrahydrate (sodium perborate tetrahydrate) calciumperoxide; peroxide complexes, urea hydrogen peroxide; superoxide salts,sodium superoxide; superoxide free radical (O₂*) dismutates; organicperoxides, hydroperoxides (ROOH), lipid hydroperoxides, artemisinin andderivatives thereof; 1-Hydroperoxycyclohexyl-1-hydroxy cyclohexylperoxide, tert-Butyl hydroperoxide; fatty acid hydroperoxides, linolenicacid hydroperoxide, arachidonic acid hydroperoxide, docosahexaenoic acidhydroperoxide, cholesterol hydroperoxide, cholesteryl linoleatehydroperoxide, trilinolein hydroperoxide, phosphatidylcholinehydroperoxide, phosphatidylethanolamine hydroperoxide;3-chloroperoxybenzoic acid, 1,1-bis(tert-butylperoxy)cyclohexane,peracetic acid, monoperoxyphthalic acid, tert-butyl peroxide,2,5,bis(tert-butylperoxy)-2,5-dimethyl-3-hexyne, cumene hydroperoxide,2,5-bis(tert-butylperoxy)-2,5-dimethylhexane, lauroyl peroxide, benzoylperoxide, dicumyl peroxide, 2,5-dihydroperoxy-2,5-dimethylhexane,tert-butyl peracetate, tert-amyl hydroperoxide, diisononanoyl peroxide,decanoyl peroxide, succinic acid peroxide, 2,4-pentanedione peroxide,di-tert-butyl peroxide, tert-butyl cumyl peroxide,bis(t-butylperoxy)-diisopropylbenzene, and1-((hydroperoxycyclohexyl)dioxy)-cyclohexanol; hydrogen peroxide,compounds containing a peroxy (peroxo) —O—O— moiety, superoxides,peroxide precursor compounds; endoperoxides, diacyl peroxides, ketoneperoxides, peroxydicarbonates, peroxyesters, dialkyl peroxides,peroxyketals, and peroxyacids.
 25. The pharmaceutical composition ofclaim 23, wherein the oxidating agent comprises hydrogen peroxide,tert-butyl hydroperoxide (TBHP), and arsenic trioxide.